Publicação
Filamentous bacteria population control: bacteriophage isolation and characterization
| Resumo: | The overgrowth of filamentous bacteria leads to worldwide operational problems in activated sludge processes in wastewater treatment plants (WWTP), the most recurring being filamentous bulking and/or filamentous foaming, both resulting in effluents with poor quality. Current control measures of filamentous bacteria are not always effective and are not sustainable and economically viable in the long run. An alternative solution can be the use of bacteriophages to control the overgrowth of filamentous bacteria. The present thesis envisaged the isolation and characterization of a lytic phage which specifically infects Sphaerotilus natans, a filamentous bacterium which causes filamentous bulking, and the evaluation of the effectiveness of the isolated phage in controlling the population of S. natans in batch assays. The growth kinetics parameters of the strain used in this thesis, S. natans DSM 6575T were calculated. Specific growth rate and the generation time of this strain were found to be 0.152 ± 0.002 h-1 and 4.560 h, respectively. The principal objective of the thesis was achieved: a lytic phage with S. natans as a host was isolated from the mixed liquor of a bench scale reactor (4 L) that accurately mimic the secondary treatment of WWTP. Phage infection on pure culture of the host strain resulted in small, uniform and clear plaques, with 1.3 ± 0.3 mm in diameter. S. natans phage belongs to the Podoviridae family, possess an icosahedral head with a size of 60 nm, along with a short tail of 20 nm length by 10 nm width. This newly isolated S. natans phage is the first ever, among those that have S. natans as host, to be classified in this family. S. natans phage did not show a lytic effect against any of the other bacteria tested. S. natans phage proved to be stable after 24 h within the temperatures of 4 °C, 21 °C, 28 °C and 40 °C and within the pH range of 5 to 11. S. natans phage possesses a latent period of 2 h and a burst size of 331.4 PFU per infected cell. The use of S. natans phage at MOI 1 proved to be the most suitable for the purpose of its application, since after 48 h results in the reduction of bacteria concentration in 0.88 logs. Results obtained indicate a possible lytic nature of the phage, a narrow host range and thermal and pH stability within the range values reported in activated sludge processes. In conclusion, the characterization performed on S. natans phage provided the results needed for the future proof of concept regarding the biocontrol of the overgrowth of S. natans by this phage, in activated sludge wastewater treatment. |
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| Autores principais: | Cardoso, Rui Manuel Amado |
| Assunto: | Bacteriophage Sphaerotilus natans Biocontrol Activated sludge Filamentous bacteria Bacteriófago Biocontrolo Lamas ativadas Bactérias filamentosas |
| Ano: | 2018 |
| 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: | The overgrowth of filamentous bacteria leads to worldwide operational problems in activated sludge processes in wastewater treatment plants (WWTP), the most recurring being filamentous bulking and/or filamentous foaming, both resulting in effluents with poor quality. Current control measures of filamentous bacteria are not always effective and are not sustainable and economically viable in the long run. An alternative solution can be the use of bacteriophages to control the overgrowth of filamentous bacteria. The present thesis envisaged the isolation and characterization of a lytic phage which specifically infects Sphaerotilus natans, a filamentous bacterium which causes filamentous bulking, and the evaluation of the effectiveness of the isolated phage in controlling the population of S. natans in batch assays. The growth kinetics parameters of the strain used in this thesis, S. natans DSM 6575T were calculated. Specific growth rate and the generation time of this strain were found to be 0.152 ± 0.002 h-1 and 4.560 h, respectively. The principal objective of the thesis was achieved: a lytic phage with S. natans as a host was isolated from the mixed liquor of a bench scale reactor (4 L) that accurately mimic the secondary treatment of WWTP. Phage infection on pure culture of the host strain resulted in small, uniform and clear plaques, with 1.3 ± 0.3 mm in diameter. S. natans phage belongs to the Podoviridae family, possess an icosahedral head with a size of 60 nm, along with a short tail of 20 nm length by 10 nm width. This newly isolated S. natans phage is the first ever, among those that have S. natans as host, to be classified in this family. S. natans phage did not show a lytic effect against any of the other bacteria tested. S. natans phage proved to be stable after 24 h within the temperatures of 4 °C, 21 °C, 28 °C and 40 °C and within the pH range of 5 to 11. S. natans phage possesses a latent period of 2 h and a burst size of 331.4 PFU per infected cell. The use of S. natans phage at MOI 1 proved to be the most suitable for the purpose of its application, since after 48 h results in the reduction of bacteria concentration in 0.88 logs. Results obtained indicate a possible lytic nature of the phage, a narrow host range and thermal and pH stability within the range values reported in activated sludge processes. In conclusion, the characterization performed on S. natans phage provided the results needed for the future proof of concept regarding the biocontrol of the overgrowth of S. natans by this phage, in activated sludge wastewater treatment. |
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