MICROCOSM EVALUATION AND METAGENOMIC ANALYSIS OF THE BIOREMEDIATION OF SOILS CONTAMINATED WITH PAHS BY MICROBIAL CONSORTIA GERMAN ALEXIS ZAFRA SIERRA INSTITUTO POLITÉCNICO NACIONAL CENTRO DE INVESTIGACIÓN EN BIOTECNOLOGÍA APLICADA MICROCOSM EVALUATION AND METAGENOMIC ANALYSIS OF THE BIOREMEDIATION OF SOILS CONTAMINATED WITH PAHS BY MICROBIAL CONSORTIA GERMAN ALEXIS ZAFRA SIERRA A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Sciences on Biotechnology November, 2014 ADVISORS: Diana V. Cortés Espinosa, PhD. Instituto Politécnico Nacional Centro de Investigación en Biotecnología Aplicada Angel E. Absalón Constantino, PhD. Instituto Politécnico Nacional Centro de Investigación en Biotecnología Aplicada THESIS COMMITTEE: Miguel Ángel Anducho Reyes, PhD. Universidad Politécnica de Pachuca Francisco José Fernández Perrino, PhD. Universidad Autónoma Metropolitana, Iztapalapa Martha D. Bibbins Martínez, PhD. Instituto Politécnico Nacional Centro de Investigación en Biotecnología Aplicada Víctor Eric López y López, PhD. Instituto Politécnico Nacional Centro de Investigación en Biotecnología Aplicada Imagination will often carry us to worlds that never were. But without it we go nowhere. Carl Sagan (1934-1996) ACKNOWLEDGEMENTS First and foremost, my greatest thanks to my wonderful family and relatives for their enduring support and encouragement all the way through. Their unconditional support since many years have given me the strength to pursue my goals and dreams, no matter where they take me. To my dear parents, my sister and nephew who have never stopped believing in me, thank you; this thesis is dedicated to you. I want to express my sincere gratitude to my principal advisor, Dr. Diana Cortés, for allowing me into her laboratory, for her guidance, valuable advice and her confidence in my work and research abilities. I also want to thank Dr. Angel Absalón for his important support and recommendations. I would also like to extent my appreciation to my thesis committee members, Dr. Martha Bibbins, Dr. Miguel A. Anducho, Dr. Eric Lopez and Dr. Francisco J. Fernandez for their recommendations, guidance, enthusiasm and encouragement. It has been a privilege having all their expertise and valuable contributions in this project. I want to express my deep gratitude to Dr. Todd D. Taylor from RIKEN-IMS for hosting me in his laboratory, making possible the analysis of metagenomes. I will be forever indebted to Todd and all the team members of the Laboratory for Integrated Bioinformatics for all the help, guidance, recommendations, funny moments and for making my stay in Japan a very formative and unforgettable experience. ありがとうございました! A very special thanks to the staff, colleagues and friends at AIRB research group at UPP for the help and support in the realization of DGGE analyses. To all my colleagues and friends at CIBA-IPN who helped me along the way, I am extremely grateful to all of you. Thank you! Finally, I gratefully acknowledge CONACYT and Instituto Politécnico Nacional for scholarships, mobility grants and financial support during my doctoral studies. LIST OF PUBLICATIONS This is a thesis by publication, in which each article form the individual chapters of the thesis. Articles are either published, accepted for publication or to be submitted for publication in peer- reviewed journals and are listed as follows: CHAPTER 2 Zafra G., Absalón A.E., Cuevas M.C., Cortés-Espinosa D.V. Isolation and selection of a highly tolerant microbial consortium with potential for PAH biodegradation from heavy crude oil- contaminated soils. Water Air and Soil Pollution 2014; 225(2):1826. DOI: 10.1007/s11270-013- 1826-4 JCR impact factor: 1.685 CHAPTER 3 Zafra G., Moreno-Montaño A., Absalón A.E., Cortés-Espinosa D.V. Degradation of Polycyclic Aromatic Hydrocarbons in soil by a tolerant strain of Trichoderma asperellum. Environmental Science and Pollution Research, 2014. In press (Epub ahead of print). DOI: 10.1007/s11356-014- 3357-y JCR impact factor: 2.757 CHAPTER 4 Zafra G., Absalón A.E., Cortés-Espinosa D.V. Morphological changes and growth of filamentous fungi in presence of high concentrations of PAHs. Accepted for publication in Brazilian Journal of Microbiology. JCR impact factor: 0.762 CHAPTER 5 Zafra G., Absalón A.E., Anducho-Reyes M.A., Cortés-Espinosa D.V. Construction of PAH- degrading Mixed Microbial Consortia by induced Selection in Soil. To be submitted to Microbial Ecology JCR impact factor: 3.118 CHAPTER 6 Zafra G., Absalón A.E., Cortés-Espinosa D.V. Selection of Optimal Culture Conditions for PAH Degradation in Soils by Microbial Consortia. To be submitted to Environmental Science and Pollution Research JCR impact factor: 2.757 CHAPTER 7 Zafra G., Absalón A.E., Taylor T.D., Cortés-Espinosa D.V. Comparative metagenomic analysis of PAH bioremediation in soil by mixed microbial consortia. To be submitted to Environmental Science & Technology JCR impact factor: 5.481 CHAPTER 8 Zafra G. & Cortés-Espinosa D.V. Degradation of aromatic hydrocarbons by Trichoderma species: an overview. (Review). To be submitted to Fungal Biology Reviews JCR impact factor: 1.362 I warrant that I have obtained, where necessary, permission from the copyright owners to use any third party copyright material reproduced in the thesis (e.g., artwork, unpublished work), or to use any of my own published work (e.g. journal articles) in which the copyright is held by another party (e.g. publisher, co-author). STATEMENT OF CONTRIBUTIONS BY OTHERS I declare that the work presented in this thesis was primarily designed, experimentally executed, interpreted and written by the first author of the individual manuscripts (German Zafra). Contributions by other authors are described in the following: CHAPTER 2 Diana V. Cortés-Espinosa provided significant contributions to the conception and design of this study. Ma. del Carmen Cuevas provided the soil samples used for microbial isolation. Diana V. Cortés-Espinosa and Angel Absalón provided critical and through revision of the manuscript. CHAPTER 3 Diana V. Cortés-Espinosa provided significant contributions to the conception and design of this study. Angélica Moreno provided technical support in the realization of enzymatic assays. Diana V. Cortés-Espinosa and Angel Absalón provided critical and through revision of the manuscript. CHAPTER 4 Diana V. Cortés-Espinosa provided contributions to the conception and design of this study. Diana V. Cortés-Espinosa and Angel Absalón provided critical revision of the manuscript. CHAPTER 5 Diana V. Cortés-Espinosa and Angel Absalón provided contributions to the conception and design of this study. Miguel A. Anducho provided logistical support for the realization of DGGE assays. Diana V. Cortés-Espinosa and Angel Absalón provided critical and through revision of the manuscript. CHAPTER 6 Diana V. Cortés-Espinosa provided significant contributions to the conception and design of this study. Miguel A. Anducho provided logistical support for the realization of DGGE assays. Diana V. Cortés-Espinosa and Angel Absalón provided critical revision of the manuscript. CHAPTER 7 Diana V. Cortés-Espinosa and Angel Absalón provided important contributions to the conception and design of this study. Todd Taylor provided facilities for computational analysis and contributed in the analysis and interpretation of metagenomic data. All coauthors provided critical revision of the manuscript. CHAPTER 7 All coauthors contributed equally to the preparation of the manuscript. CONFERENCE PRESENTATIONS Zafra G., Absalón A.E., Cortés-Espinosa D. Identification of Highly Tolerant Fungal Isolates to Polycyclic Aromatic Hydrocarbons. XV National Congress of Biotechnology and Bioengineering. Cancún, México. June 2013 (Poster presentation). Zafra G., Absalón A.E., Cortés-Espinosa D. Determination of Bacterial Tolerance to High Concentrations of PAHs. Second International Symposium on Bioremediation and Sustainable Environmental Technologies. Jacksonville, USA. June 2013. (Poster presentation). In extenso paper published in: Proceedings of the Second International Symposium on Bioremediation and Sustainable Environmental Technologies. 2013. A-13, pp. 1-6. ISBN 978-0- 9819730-7-4 Zafra G., Absalón A.E., Taylor T.D., Cortés-Espinosa D. Functional metagenomic analysis of the bioremediation of a hydrocarbon-polluted soil with a mixed microbial consortium. 4th International Symposium on Environmental Biotechnology and Engineering. Mexico City, Mexico. September 2014. (Oral presentation). In: Environmental Biotechnology and Engineering - 2014. ISBN 978-607-9023-24-9. Zafra G., Absalón A.E., Taylor T.D., Cortés-Espinosa D. Microbial diversity shifts during the bioremediation of a heavy crude oil-contaminated soil at microcosm. 1st Biotechnology World Symposium. Tlaxcala, Mexico. October 2014 (Poster presentation). In: Journal of Chemical, Biological and Physical Sciences. ISSN 2249-1929. Zafra G., Absalón A.E., Anducho-Reyes M.A., Cortés-Espinosa D. Selection of Optimal Culture Conditions for PAH Removal in Soils by Mixed Microbial Consortia. 2014-Contaminated Site Management: Sustainable Remediation & Management of Soil, Sediment and Water. San Diego, USA. November 2014 (Oral presentation). TABLE OF CONTENTS . TABLE OF CONTENTS ABSTRACT .............................................................................................................................................. iii RESUMEN ................................................................................................................................................