Micropollutant bioremoval in wastewater treatment systems: from microbial population structure to function Bárbara Fonseca de Almeida Insert here an image with rounded corners Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, February, 2013 Micropollutant bioremoval in wastewater treatment Bárbara Fonseca Oeiras, February, 2013 systems: from microbial populaon structure to func6on de Almeida Micropollutant bioremoval in wastewater treatment systems: from microbial population structure to function Bárbara Fonseca de Almeida Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, February, 2013 i Financial Support from Fundação para a Ciência e a Tecnologia (FCT) – PhD grant - SFRH/BD/47748/2008. iii To my family v Acknowledgements I would first like to express my gratitude to Doctor Teresa Crespo and Doctor Gilda Carvalho, who are not only supervisors but also dear friends. I am forever grateful for all their support, confidence and guidance during these four years. I would also like to thank my thesis committee, Professor Olga Nunes and Professor João Paulo Noronha, who provided encouraging and constructive feedback. I am very thankful to Professor Jeppe Lund Nielsen for hosting my visit to the Environmental Engineering Group, at the Aalborg University, Denmark; and to Professor Olga Nunes for hosting my visit to the DEQ/FEUP. I was kindly welcomed in both facilities, and learned many techniques and approaches that contributed to my knowledge and this thesis. I want to acknowledge António Ferreira, Mário Patrício and Susana Tenedório at IBET Analytical Services Unit, who helped make this work possible. I wish to thank the co-authors of the publications included in this thesis. This work would not have been possible without their help. vii To the many colleagues and friends at ITQB, IBET, AAU and FEUP who made these places very special and remarkable. I will always remember you. I would like to thank my many friends for their invaluable support and friendship. In particular, Carmo, Dulce, Ihab, Manel, Vang, Charlotte, Sofia and Hugo, with whom I shared most of the ups and downs of this thesis. Thank you for always being present. To my family. Especially to my “mams”, to my “paps”, my sisters Kéu and Carol and my “vó”, for their unconditional love, patience and support. Their belief in me has always been strong. Last of all, my gratitude and love must fall upon my most patient companion, Vinny, who has always supported me. Thank you for always showing me my true self. viii Abstract The continuous release of micropollutants into receiving waters due to insufficient elimination from wastewater treatment plants (WWTP) raises global concerns regarding their potential risks to the environment and human health. Most treatment plants employ biological systems to degrade organic matter due to economic and environmental advantages but they are not designed to select microbial consortia that possess the necessary enzymes to remove micropollutants. Therefore, it is important to characterise the microbial populations in treatment systems in order to optimise the elimination of these compounds from WWTP. This work characterises the performance, metabolism and identity of the microbial populations involved in the degradation of target micropollutants, namely two pharmaceutical compounds and a heavy metal. The main factors influencing the performance of activated sludge during the degradation of ibuprofen and ketoprofen are described in this study using mathematical models. The model accurately predicted the degradation of both compounds, which was found to be a function of biomass composition and substrate loading rate. From one of the characterised activated sludge, 12 microorganisms were isolated that were possibly involved in the ibuprofen and ketoprofen biodegradation. One of the isolates was characterised via another model, where ibuprofen degradation performance was dependent on both biomass growth and initial substrate concentration. This isolate with unique ix phenotypical and molecular characteristics has been classified as a new species (Patulibacter medicamentivorans). Furthermore, the metabolic aspects of ibuprofen degradation by Patulibacter medicamentivorans were investigated through quantitative proteomics using a metabolic labelling strategy. The whole genome of this microorganism was sequenced to provide a species-specific protein platform for optimal protein identification. Several proteins were identified that are possibly involved in the degradation of ibuprofen and that will enable further studies aiming at characterising the mechanisms of microbial community development and dynamics in engineered systems. An example of such an application was conducted in this thesis to assess the evolution of mercury reducing microbial communities developed under different operating conditions. The genetic (16S rRNA gene) and functional (merA gene) characteristics of the mercury-reducing cultures enriched with different carbon sources (glucose, ethanol or acetate) were investigated. The choice of the carbon source was shown to have profound effects on both the phylogenetic development of microbial cultures and mercury reducing performance. This thesis represents a step towards a better understanding of the microbial communities involved in the degradation of important micropollutants. This knowledge can be applied in the future for improved design and operation of water and wastewater treatment systems in order to enhance the removal of these compounds. x Sumário A emissão constante de micropoluentes em meios aquáticos, devido à eliminação ineficiente destes compostos nas estações de tratamento de água residual (ETAR), tem suscitado preocupações a nível mundial a respeito de riscos adversos potenciais para o meio ambiente e saúde humana. A maioria das ETARs, utilizam processos biológicos para remover contaminantes orgânicos da água residual por serem processos vantajosos em termos económicos e ambientais. No entanto, estes sistemas não estão projectados para seleccionar consórcios microbianos que possuam as enzimas necessárias para remover micropoluentes, levando à eliminação ineficiente destes compostos. Perante este fato, torna-se importante caracterizar as populações microbianas em sistemas de tratamento, a fim de optimizar a eliminação destes compostos nas ETARs. O trabalho apresentado nesta tese caracteriza o desempenho, o metabolismo e a identidade das populações microbianas envolvidas na degradação de dois compostos farmacêuticos (ibuprofeno e cetoprofeno) e de um metal pesado (mercúrio). Os principais factores que influenciam o desempenho de lamas activadas durante a degradação do ibuprofeno e do cetoprofeno foram descritos neste estudo através de modelação matemática. O modelo matemático, previu com precisão a degradação de ambos os compostos, em função da composição de biomassa e de concentração inicial de substrato. Doze microrganismos, potencialmente envolvidos na biodegradação do ibuprofeno e cetoprofeno, foram isolados de uma das lamas activadas previamente caracterizada pelo modelo matemático. A degradação de ibuprofeno por um dos isolados foi descrita por outro modelo xi matemático, em função do crescimento de biomassa e da concentração inicial de substrato. Este isolado, com características fenotípicas e moleculares exclusivas foi classificado como uma espécie nova, Patulibacter medicamentivorans. Adicionalmente, os aspectos metabólicos de degradação do ibuprofeno por Patulibacter medicamentivorans, foram investigados por proteómica quantitativa usando uma estratégia de marcação metabólica. O genoma deste microrganismo foi sequenciado e usado como uma plataforma de identificação proteica específica para as proteínas desta espécie. Várias proteínas foram induzidas na presença de ibuprofeno, que provavelmente estarão envolvidas na biodegradação deste composto. A identificação destas proteínas permitirá elucidar os mecanismos de degradação, e seguir a actividade e a dinâmica de desenvolvimento das comunidades microbianas em bioreactores. Um exemplo desta aplicação foi realizada nesta tese para avaliar a evolução das comunidades microbianas reductoras de mercúrio, desenvolvidas sob diferentes condições de operação. As características genéticas (gene rRNA 16S) e funcionais (gene merA) de culturas reductoras de mercúrio enriquecidas na presença de diferentes fontes de carbono (glicose, etanol ou acetato) foram investigadas. A escolha da fonte de carbono demonstrou afectar a selecção e a filogenética das culturas microbianas, bem como a sua capacidade de redução do mercúrio. Esta tese representa um avanço no sentido de uma melhor compreensão das comunidades microbianas envolvidas na degradação de importantes micropoluentes. No futuro, este conhecimento pode ser aplicado na concepção e operação de sistemas de tratamento de águas residuais, com o objectivo de aperfeiçoar a remoção destes compostos e minimizar o seu impacto ambiental. xii List of Publications List of Publications Almeida, B., Oehmen, A., Marques, R., Brito, D., Carvalho, G., Barreto Crespo, M.T., 2012. Modelling the biodegradation of non-steroidal anti-inflammatory drugs (NSAIDs) by activated sludge and a pure culture. Submitted to J. Hazard. Mater. Almeida, B., Vaz-Moreira, I., Schumann, P., Nunes, O.C., Carvalho, G., Barreto Crespo, M.T., 2012. Patulibacter medicamentivorans sp.