Publicação
The role of fungi in drinking water biofilm formation and behavior
| Resumo: | 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<0,05) compared to the static and 30 rpm conditions, meaning that high rotations might influence the fungus growth and metabolic activity. In this condition, there was an increase in biomass and metabolic activity, suggesting that the high rotations had a positive influence. Comparative to the nutrient’s concentration, four different conditions were evaluated (synthetic tap water, ¼ R2B, ½ R2B, and R2B medium). After three days of incubation, that was observed that STW caused significant differences (ρ<0,05) compared to the others, meaning that the oligotrophic environment influences the fungus growth and metabolic activity negatively. The exposure to chlorine was studied under 5 conditions (without chlorine, 2,4 ppm, 6,03 ppm, 12,06 ppm, 24,12 ppm) and revealed no significant impact of the chlorine levels in biofilm formation and activity, suggesting the fungi resistance to chlorine. For the interkingdom factor, a strain of Stenotrophomonas maltophilia was used. Three assays were performed (Fungus and bacterium alone and associated). The results revealed that microbial association affected biofilm formation and activity, showing a decrease in biomass and metabolic activity compared to the fungi assay alone. Lastly, the species identification of two fungi used in this work was executed as well. The Alternaria sp. and Mucor sp. were the fungi that were not previously identified until the species group. For this identification, the fungi were incubated in R2B medium, and the DNA was extracted. After running some tests, such as the NanoDrop, the electrophoresis gel of the sample, and PCR cycle, the Fungi DNA was sequenced. Then, a phylogenetic tree was created based on the genetic sequences, with the same genetic marker (ITS), of different fungi species related to the samples. Thus, the phylogeny tree of the Mucor seems to direct the Mucor sp. toward the Mucor plumbeus and the phylogeny tree of the Alternaria put the sample in the Alternata section. |
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| Autores principais: | Ferreira, Simão Pedro de Duarte |
| Assunto: | Engenharia e Tecnologia |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | 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<0,05) compared to the static and 30 rpm conditions, meaning that high rotations might influence the fungus growth and metabolic activity. In this condition, there was an increase in biomass and metabolic activity, suggesting that the high rotations had a positive influence. Comparative to the nutrient’s concentration, four different conditions were evaluated (synthetic tap water, ¼ R2B, ½ R2B, and R2B medium). After three days of incubation, that was observed that STW caused significant differences (ρ<0,05) compared to the others, meaning that the oligotrophic environment influences the fungus growth and metabolic activity negatively. The exposure to chlorine was studied under 5 conditions (without chlorine, 2,4 ppm, 6,03 ppm, 12,06 ppm, 24,12 ppm) and revealed no significant impact of the chlorine levels in biofilm formation and activity, suggesting the fungi resistance to chlorine. For the interkingdom factor, a strain of Stenotrophomonas maltophilia was used. Three assays were performed (Fungus and bacterium alone and associated). The results revealed that microbial association affected biofilm formation and activity, showing a decrease in biomass and metabolic activity compared to the fungi assay alone. Lastly, the species identification of two fungi used in this work was executed as well. The Alternaria sp. and Mucor sp. were the fungi that were not previously identified until the species group. For this identification, the fungi were incubated in R2B medium, and the DNA was extracted. After running some tests, such as the NanoDrop, the electrophoresis gel of the sample, and PCR cycle, the Fungi DNA was sequenced. Then, a phylogenetic tree was created based on the genetic sequences, with the same genetic marker (ITS), of different fungi species related to the samples. Thus, the phylogeny tree of the Mucor seems to direct the Mucor sp. toward the Mucor plumbeus and the phylogeny tree of the Alternaria put the sample in the Alternata section. |
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