Screening hydrolase-producing environmental bacteria towards their application in bioremediation Maria Cristina Lopes Matias Thesis to obtain the Master of Science Degree in Microbiology Supervisor: Professor Rogério Paulo de Andrade Tenreiro Co-supervisor: Professor Nuno Gonçalo Pereira Mira Examination Committee Chairperson: Professor Isabel Maria de Sá Correia Leite de Almeida Supervisor: Professor Rogério Paulo de Andrade Tenreiro Member of the Committee: Professor Ana Cristina Anjinho Madeira Viegas October 2019 Declaration I declare that this document is an original work of my own authorship and that it fulfills all the requirements of the Code of Conduct and Good Practices of the Universidade de Lisboa. i Preface The work presented in this thesis was performed at Lab Bugworkers | M&B-BioISI, Faculty of Sciences of the University of Lisbon (Lisbon, Portugal), during the period from September 2017 to October 2019, under the supervision of Professor Rogério Tenreiro. The thesis was co-supervised at Instituto Superior Técnico (Lisbon, Portugal) by Professor Nuno Mira. ii Acknowledgments This thesis is the end result of the work developed within the Microbiology & Biotechnology Group of Biosystems and Integrative Sciences Institute (M&B-BioISI), at Lab Bugworkers | M&B-BioISI, located in the Innovation Centre from the Faculty of Sciences of the University of Lisbon, Tec Labs. It would not have been successfully achieved without the help and collaboration of several people, to whom I would like to express my sincere gratitude. First, and foremost, I would like to thank my supervisor, Professor Rogério Tenreiro, for challenging me with this project. I have to thank him for the patience for my endless questions, for letting me error along the way (and learn with those errors), and for all the ideas to improve my work. I would also like to thank Professor Nuno Mira, my internal supervisor at Instituto Superior Técnico, for his advice. This work was based on a collection of man-made environmental naturally occurring bacteria originated from a collaboration between the Lab Bugworkers | M&B-BioISI and the company BioTask, Biotechnology Solutions, Lda., so I would like to thank BioTask for the courtesy of allowing me the use of the BBC collection in my work. In this context, a special thanks to Pedro Teixeira, who established the BBC collection in the course of his PhD, for his support, availability, and for all that I learned from him. To Professor Ana Tenreiro, my thanks, for her reassuring presence, ideas, and for helping me with some of the most technical aspects of my work. To Filipa Silva, Chief Laboratory Officer of the Lab Bugworkers, for her availability, and for all that I learned from her; to her, and to the laboratory technician Francisca Moreira, my thanks for keeping the lab working efficiently. To Professor Lisete Fernandes, for the after-hours talks. To my colleagues in the Lab, with a special regard to my fellow colleagues doing their master thesis, for their companionship, and for all their support. I would also like to thank my family. Without them, I would not have been here. A very special thanks to my sister, for the patience, and for drawing my attention for the little details on my ongoing thesis, even if she does not have a biological sciences background. Last, but not least, I would like to thank Alexandre Calapez, for all the incentive and support when I decided to go back to school, and throughout all this period. Also, I am very grateful for all the long hours reviewing my thesis, and for all the advice that came to improve it. iii Abstract Hydrolases are an important type of enzymes, playing a valuable role in the turnover of nutrients, by promoting the depolymerization of macromolecules. Hydrolases are very potent catalysts, frequently secreted to the surrounding medium, most do not require cofactors, characteristics that make them desirable for biotechnological applications, in particular for environment protection, where hydrolase-producing organisms can be used in the removal of contaminants, in a process called bioremediation. In this work, a set of 25 strains, selected from a collection of man-made environmental naturally occurring bacteria, was phenotypically characterized (laboratory routine tests, and the utilization of 23 substrates as sole carbon source), screened for 12 hydrolase activities (carboxylic ester hydrolases, deoxyribonucleases, eight glycosidases, peptidases, and ureases), and identified, by 16S rRNA gene sequencing and phylogenetic analysis. From this set, two subsets of strains, one able to degrade urea, and one able to degrade cellulose, were selected for further assays, regarding their potential application in the biological treatment of wastewaters. The most promising candidate was an Enterobacter sp. strain, isolated from a petrochemical wastewater treatment plant, found to be able to grow with urea as sole nitrogen source, and capable of urea and ammonium removal in synthetic wastewater. The strain is also able to grow in nutritious medium with up to 6.66% w/v of urea, as well as with up to 6.00% w/v of ammonium chloride. Such remarkable features pave the way for the future use of this strain as an asset for treatment of urea or ammonium rich wastewaters. Keywords Hydrolase screening, bioremediation, wastewater treatment, urea, ammonium, cellulose. iv Resumo As hidrolases são um tipo de enzimas importante porque, ao promoverem a degradação de macromoléculas, desempenham um papel crucial no ciclo dos nutrientes. As hidrolases são catalisadores extremamente potentes, são geralmente excretadas para o exterior, e a maioria não requer cofatores, características que as tornam desejáveis para aplicações biotecnológicas, em particular no campo da proteção ambiental, onde organismos produtores de hidrolases podem ser utilizados na remoção de contaminantes. Este processo é designado por biorremediação. Neste estudo, um grupo de 25 estirpes, selecionado a partir de uma coleção de bactérias de ocorrência natural em ambientes artificiais, foi alvo de caracterização fenotípica (testes de rotina laboratorial e utilização de 23 substratos diferentes como fonte única de carbono) e de prospeção de 12 atividades hidrolíticas (carboxil éster hidrolases, deoxirribonucleases, oito glicosidases, peptidases e ureases). As estirpes foram posteriormente identificadas a partir da sequenciação do gene 16S rRNA e de análise filogenética. Deste grupo de 25 estirpes foram depois selecionados dois subgrupos para estudar a sua potencial aplicação no tratamento biológico de águas residuais: um com estirpes produtoras de ureases e um com estirpes capazes de hidrolisar celulose. O candidato mais promissor encontrado foi uma estirpe do género Enterobacter, isolada de uma estação de tratamento de águas de uma instalação petroquímica. A estirpe é capaz de crescer com ureia como fonte única de azoto, e também capaz de remover ureia e amoníaco de água residual sintética. É ainda capaz de crescer em meio nutritivo suplementado com até 6.66% m/v de ureia, e em meio nutritivo com até 6.00% m/v de cloreto de amónio. Com estas características esta estirpe de Enterobacter pode vir a constituir um novo recurso no tratamento de águas residuais ricas em ureia ou amónia. Palavras-chave Prospeção de hidrolases; biorremediação; tratamento de águas residuais; ureia, amónia; celulose. v Table of Contents Declaration ...............................................................................................................................................................i Preface .................................................................................................................................................................... ii Acknowledgments ................................................................................................................................................. iii Abstract .................................................................................................................................................................. iv Resumo .................................................................................................................................................................. v Table of Contents .................................................................................................................................................. vi List of Figures ........................................................................................................................................................ x List of Tables .......................................................................................................................................................... xi List of Acronyms & Abbreviations ..................................................................................................................... xiii 1. Introduction ...................................................................................................................................................... 1 1.1. Environmental sustainability, green chemistry, and catalysis ............................................................... 1 1.2. Enzymes and biotechnological applications – an overview ................................................................... 1 1.3. Enzyme classification ................................................................................................................................ 2 1.3.1. International Union of Biochemistry