Adaptation Studies and Proteomic Analysis of Carboxidotrophic Methanogens
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Adaptation Studies and Proteomic Analysis of Carboxidotrophic Methanogens Ricardo Afonso Gonçalves Pereira Thesis to obtain the Master of Science Degree in Biological Engineering Supervisors: Prof. Diana Zita Machado de Sousa Prof. Miguel Nobre Parreira Cacho Teixeira Examination Committee Chairperson: Prof. Arsénio do Carmo Sales Mendes Fialho Supervisor: Prof. Miguel Nobre Parreira Cacho Teixeira Members of the Committee: Prof. Nuno Gonçalo Pereira Mira December 2014 Page | ii Abstract Carbon monoxide (CO) is generated by a variety of natural and anthropogenic processes. Nevertheless, CO is only present at trace amounts in the atmosphere, which is partly due to its utilisation by a wide range of diverse prokaryotes, both aerobic and anaerobic. Organisms able to autotrophically grow on CO, i.e. to use CO as carbon and electron source, are designated carboxydotrophs. Anaerobic carboxydotrophs are particularly interesting for biotechnological applications because they can produce added-value compounds, such as hydrogen, methane, fatty- acids and alcohols. CO is one of the main compounds of synthesis gas (or syngas) - product resulting from the gasification of carbonaceous sources (e.g. lignocellulosic biomass and wastes). In this way, syngas can be used by carboxydotrophs as a sustainable source for the production of chemicals and/or biofuels. The present thesis focused on the study of methanogens for the production of methane from CO and CO/H2 mixtures as sole substrate/electron donor. Several methanogenic species were tested for their ability to metabolise CO and/or CO/H2 and the mechanisms and requirements for CO tolerance and oxidation were studied. The physiological response of these archaea to the presence of CO was detailed and the previously unreported carboxidotrophic ability for Methanothermobacter marburgensis was demonstrated. Furthermore, proteomic analysis of the CO-metabolizing capability of this organism was performed. Keywords: Carbon Monoxide; Synthesis Gas; Methanogenesis; Adaptation; Proteomics; Methanothermobacter marburgensis Page | iii Resumo O monóxido de carbono (CO) é produzido por uma variedade de processos naturais e antropogénicos, estando, no entanto, presente em quantidades vestigiais na atmosfera, o que é em parte devido à sua utilização por uma ampla gama de diversos procariotas, tanto aeróbios como anaeróbicos. Os organismos capazes de crescer autotroficamente em CO, i.e. de usá-lo como fonte de carbono e electrões, são designados carboxidotróficos. Os carboxidotróficos anaeróbios possuem especial interesse em aplicações biotecnológicas devido ao seu potencial de produção de compostos com valor acrescentado, tais como hidrogénio, metano, ácidos gordos e álcoois. O CO é um dos principais compostos do gás de síntese (ou syngas) - produto resultante da gasificação de fontes carboníferas (e.g. biomassa lignocelulósica e resíduos) e, deste modo, pode ser usado por carboxidotróficos como uma fonte sustentável para a produção de químicos e/ou biocombustíveis. O foco da presente tese é o estudo de metanogénicos para a produção de metano a partir de CO e misturas de CO/H2 como único substrato/dador de electrões. Várias espécies metanogénicas foram testadas quanto à sua capacidade de metabolizar CO e/ou CO/H2 e os mecanismos e requisitos necessários à tolerância e oxidação de CO foram também estudados. A resposta fisiológica destes archaea à presença de CO foi detalhada e foi pela primeira vez demonstrada a capacidade carboxidotrófica de Methanothermobacter marburgensis. Além disso, foi efetuada a análise proteómica da capacidade metabolizante de CO deste organismo. Palavras-Chave: Monóxido de Carbono; Gas de Síntese; Metanogenese; Adaptação; Proteómica; Methanothermobacter marburgensis Page | iv Aknowledgements First and foremost I’d like to offer my gratitude to Prof. Diana Sousa and Prof. Fons Stans for giving me the opportunity to perform this work at the Microbial Physiology group in the Microbiology Department of Wageningen University. Their invaluable guidance tempered by the willingness to allow me to venture out on my own, made for a great learning experience. I’d particularly like to thank Diana for her kind words and incentive in the rougher moments of my stay. I’d also like to give a special aknowledgement to Ana Luísa Pereira who patiently taught me the basics of anaerobic culturing and shared a lot of great moments in the lab in the first few months. Other special thanks go to the ever present technitians, particularly Ton van Gelder, whose teachings and concern for my safety at all times I will not forget. In my office I had a wonderful environment of support and friendship in Ahmad Khadem, Anna Florentino, Lara Paulo and Vicente Nunez. Thank you for all for your sage advice and friendship. As we would say: “thanks for listening”. I also cannot forget the wonderful talks (scientific and otherwise) with my co-workers. Particularly Irene Sanchez-Andrea, Peer Timmers, Michael Visser and Martijn Diender were always available to discuss my scientific troubles which I appreciated. Finally I’d like to thank the entire MicFys group for making me feel welcomed and a part of the family. This study has been funded by FEDER, through the Operational Programme Thematic Factors of Competitiveness - COMPETE, and by Portuguese funds, through the Portuguese Foundation for Science and Technology (FCT), in the frame of the project FCOMP-01-0124-FEDER- 027894 - “SYN2value - Syngas bio-upgrading to fuels and chemicals”. Page | v Page | vi Contents Abstract ...................................................................................................................................... iii Resumo ....................................................................................................................................... iv Aknowledgements ....................................................................................................................... v Contents .................................................................................................................................... vii List of Figures............................................................................................................................... x List of Equations ......................................................................................................................... xi List of Tables .............................................................................................................................. xii List of Acronyms ....................................................................................................................... xiii 1 Introduction ......................................................................................................................... 1 1.1 Research motivation and background......................................................................... 1 1.2 Syngas and microbial routes for syngas/CO conversion to fuels and chemicals ........ 2 1.3 Other applications of CO-oxidizing microorganisms ................................................... 5 1.4 Microbial metabolism of carbon monoxide ................................................................ 6 1.4.1 Hydrogenogenic CO-oxidizers .............................................................................. 10 1.4.2 Acetogenic bacteria .............................................................................................. 11 1.4.3 Sulphate-reducing bacteria .................................................................................. 12 1.4.4 Methanogens ....................................................................................................... 13 1.5 CO toxicity towards microorganisms......................................................................... 16 1.6 Biochemistry of CO oxidation .................................................................................... 17 1.7 Genomic and proteomic insights .............................................................................. 18 1.7.1 Genomics .............................................................................................................. 18 1.7.2 Transcriptomics .................................................................................................... 19 1.7.3 Proteomics ........................................................................................................... 19 2 Materials and methods ..................................................................................................... 21 2.1 Comparative genomic analysys for selection of candidates ..................................... 21 2.2 Source of microorganisms ......................................................................................... 21 Page | vii 2.3 Medium composition and cultivation ....................................................................... 21 2.4 Adaptation studies .................................................................................................... 23 2.5 Microscopy ................................................................................................................ 23 2.6 Analytical techniques ................................................................................................ 24 2.6.1 GC measurements ................................................................................................ 24 2.6.2 HPLC measurements ...........................................................................................