Invitation for the public defense of my thesis Physiology and biochemistry of aromatic hydrocarbon-degrading of aromatic and biochemistry Physiology bacteria that use chlorate and/or nitrate as electron acceptor as electron nitrate and/or use chlorate that bacteria Physiology and biochemistry Physiology and biochemistry of aromatic hydrocarbon-degrading of aromatic hydrocarbon- degrading bacteria that bacteria that use chlorate and/or nitrate as electron acceptor use chlorate and/or nitrate as electron acceptor The public defense of my thesis will take place in the Aula of Wageningen University (Generall Faulkesweg 1, Wageningen) on December 18 2013 at 4:00 pm. This defense is followed by a reception in Café Carré (Vijzelstraat 2, Wageningen). Margreet J. Oosterkamp J. Margreet Paranimphs Ton van Gelder ([email protected]) Aura Widjaja Margreet J. Oosterkamp ([email protected]) Marjet Oosterkamp (911 W Springfield Ave Apt 19, Urbana, IL 61801, USA; [email protected]) Omslag met flap_MJOosterkamp.indd 1 25-11-2013 5:58:31 Physiology and biochemistry of aromatic hydrocarbon-degrading bacteria that use chlorate and/or nitrate as electron acceptor Margreet J. Oosterkamp Thesis-MJOosterkamp.indd 1 25-11-2013 6:42:09 Thesis committee Thesis supervisor Prof. dr. ir. A. J. M. Stams Personal Chair at the Laboratory of Microbiology Wageningen University Thesis co-supervisors Dr. C. M. Plugge Assistant Professor at the Laboratory of Microbiology Wageningen University Dr. P. J. Schaap Assistant Professor at the Laboratory of Systems and Synthetic Biology Wageningen University Other members Prof. dr. L. Dijkhuizen, University of Groningen Prof. dr. H. J. Laanbroek, University of Utrecht Prof. dr. ir. D. Springael, Catholic University of Leuven Prof. dr. S. C. de Vries, Wageningen University This project was conducted under the auspices of the graduate school SENSE (Socio-Economic and Natural Sciences of the Environment) Thesis-MJOosterkamp.indd 2 25-11-2013 6:42:09 Physiology and biochemistry of aromatic hydrocarbon-degrading bacteria that use chlorate and/or nitrate as electron acceptor Margreet J. Oosterkamp Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday December 18 2013 at 4:00 p.m. in the Aula of Wageningen University Thesis-MJOosterkamp.indd 3 25-11-2013 6:42:09 Margreet J. Oosterkamp Physiology and biochemistry of aromatic hydrocarbon-degrading bacteria that use chlorate and/or nitrate as electron acceptor 191 pages. Thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in English and Dutch ISBN 978-94-6173-777-9 Thesis-MJOosterkamp.indd 4 25-11-2013 6:42:10 Dedicated to my parents ‘Je kunt het altijd proberen (You can always try)’ Thesis-MJOosterkamp.indd 5 25-11-2013 6:42:10 Preface In situ bioremediation by using naturally occurring or introduced microorganisms is a very attractive option for remediation of anoxic polluted soil sites. Often the appropriate conditions for microorganisms need to be created and the introduction of electron acceptors is required. Oxygen can be introduced by aeration, however, this is difficult to achieve and very costly. We focused on the use of the alternative electron acceptors nitrate and chlorate. Nitrate and chlorate are more soluble than oxygen and easy to introduce in soil via the groundwater. Some microorganisms are known to degrade soil pollutants with nitrate and/or chlorate. Nitrate-reduction is used as a method for bioremediation of anaerobic polluted soil sites. Degradation of aromatic hydrocarbons is known to involve anaerobic pathways. Interestingly, an alternative nitrate reduction pathway in which oxygen is produced was found. This light-independent in situ oxygen production offers innovative possibilities for bioremediation of anaerobic soils polluted with aromatic hydrocarbons, for example. Biochemical routes for the degradation of such hydrocarbons require mono- and dioxygenases. These enzymes cannot function under anaerobic conditions. Because of the production of oxygen from an alternative nitrate reduction pathway, oxygenases could function. Similarly, oxygen is produced when chlorate is the electron acceptor. Laboratory experiments and additional tests in the field showed that chlorate-enhanced anaerobic benzene degradation with chlorate is possible. A stable enrichment that degraded benzene with chlorate at comparable high rates as reported for aerobic benzene-degrading cultures and at 10 to 100 times higher rates than reported for anaerobic benzene-degrading cultures was obtained. One of the bacteria from this enrichment, a novel Alicycliphilus strain, has been obtained in pure culture. This bacterium degrades benzene and some other aromatic hydrocarbons with chlorate as electron acceptor, but not with nitrate as electron acceptor. The general aim of this project was to get insight into the physiological and biochemical properties of this bacterium and of other aromatic hydrocarbon degrading bacteria that use nitrate and/or chlorate as electron acceptor. Thesis-MJOosterkamp.indd 6 25-11-2013 6:42:10 Contents Page Chapter 1 General introduction 8 Chapter 2 Genome analysis and physiological comparison of 23 T Alicycliphilus denitrificans strains BC and K601 Chapter 3 Metabolic response of Alicycliphilus denitrificans strain BC 43 toward electron acceptor variation Chapter 4 Chlorate-dependent transposon-mediated modification and 62 deletion of narG in Alicycliphilus denitrificans strain BC Chapter 5 Proteomics of nitrate-dependent acetone degradation by 74 Alicycliphilus denitrificans strain BC Chapter 6 Isolation and characterization of the benzene-degrading 87 Pseudomonas stutzeri strain BN Chapter 7 Genome-based metabolic potentials of the anaerobic, 108 monoaromatic compound-degrading bacteriumGeorgfuchsia toluolica strain G5G6T Chapter 8 Proteome and genome analysis of Pseudomonas 127 chloritidismutans AW-1T that oxidizes n-decane with chlorate or oxygen as electron acceptor Chapter 9 General discussion 142 Thesis summary 153 Samenvatting proefschrift 158 References 163 Publications of the author 180 About the author 182 Acknowledgements 184 Certificate of educational PhD programme 188 Thesis-MJOosterkamp.indd 7 25-11-2013 6:42:10 Chapter 1 General introduction 8 Thesis-MJOosterkamp.indd 8 25-11-2013 6:42:10 Author and affiliations Margreet J. Oosterkamp1(a) 1 Wageningen University, Agrotechnology and food sciences group, Laboratory of microbiology, Dreijenplein 10, 6703HB, Wageningen, The Netherlands (a) Present address: Department of Animal Sciences, Energy Biosciences Institute, Institute of Genomic Biology, University of Illinois at Urbana-Champaign, 1207 W Gregory Drive, Urbana, Illinois 61801, USA. Keywords Perchlorate-reducing bacteria, chlorate-reducing bacteria, nitrate-reducing bacteria, chlorate reductase, nitrate reductase, chlorite dismutase Manuscript status Part of this thesis chapter has been published (Oosterkamp, Mehboob et al. 2011) 9 Thesis-MJOosterkamp.indd 9 25-11-2013 6:42:10 Abstract Bacteria that grow with aromatic and aliphatic hydrocarbons and use perchlorate, chlorate and/or nitrate as electron acceptor can be applied for bioremediation of soils polluted with aromatic and aliphatic hydrocarbons. Perchlorate, chlorate and nitrate are more soluble than oxygen and can reach anaerobic soil sites more readily than oxygen. Besides the beneficial effect of the addition of an electron acceptor, (per)chlorate-reducing bacteria possess chlorite dismutase that forms oxygen. This oxygen can be used to degrade aromatic and aliphatic hydrocarbons using aerobic pathways. Aerobic degradation of these compounds is a faster process than anaerobic degradation. (Per)chlorate- and nitrate-reducing microorganisms are present in various ecosystems and interact with each other. The nitrate reduction pathway has been studied in more detail than the perchlorate and chlorate reduction pathways. The nitrate reductase of the NarGHI-type, that converts nitrate to nitrite, can also convert chlorate to chlorite. Furthermore, the subunits of NarGHI-type nitrate reductase and of chlorate reductase are structurally very similar. To get more insight in the metabolism of aromatic and aliphatic hydrocarbon degrading bacteria that can use nitrate and/or chlorate as electron acceptor, further studies are needed. In this introduction chapter the research described in this thesis is defined. The background information summarized above is provided to increase understanding of the research described in this thesis and of the need to do this research. 10 Thesis-MJOosterkamp.indd 10 25-11-2013 6:42:10 Introduction Compared to cost-intensive chemical and physical methods for the clean-up of soil sites polluted with aromatic and aliphatic hydrocarbons, methods using naturally occurring or introduced microorganisms are an attractive option. Recent advances in genomics and proteomics allow more detailed research on such microorganisms (Zhao and Poh 2008). Optimal conditions for pollutant-degrading microorganisms often need to be created. For aromatic and aliphatic hydrocarbon-polluted anaerobic soils the introduction of oxygen via aeration may be used. However, aeration of anaerobic soils is difficult to achieve and very costly. An effective alternative is the use of chlorate or nitrate as terminal electron acceptors. Unlike