Microbial Degradation of Acetaldehyde in Freshwater, Estuarine and Marine Environments Phillip Robert Kenneth Pichon A thesis submitted for the degree of Doctor of Philosophy in Microbiology School of Life Sciences University of Essex October 2020 This thesis is dedicated to the memory of my mum, Elizabeth “Taylor” Pichon Acknowledgements I would like to begin by thanking my supervisors Professor Terry McGenity, Dr Boyd McKew, Dr Joanna Dixon, and Professor J. Colin Murrell, all of whom have provided invaluable guidance and support throughout this project. Their advice and encouragement have been greatly appreciated during the last four years and I am grateful for all of their contributions to this research. I would also like to thank Dr Michael Steinke for his continued support and insightful discussions during this project. Special thanks also go to Dr Metodi Metodiev and Dr Gergana Metodieva for their help with sample preparation and initial data analysis during the proteomics experiment. I would especially like to thank Farid Benyahia and Tania Cresswell-Maynard for their technical support with a variety of molecular methods, and John Green for his help and expertise in gas chromatography. I would also like to thank all of my friends and colleagues who have worked with me in Labs 5.28 and 5.32 during the last four years. My thanks also to NERC for funding this project and to the EnvEast DTP for providing a range of valuable training opportunities. To my Dad and Roisin, your support and encouragement have helped me through the toughest parts of my project. My love and thanks to you both. Finally, I wish to thank my partner, Beth. I could not have finished my PhD without your love and support. My decision to eat that sloe berry in Flatford was probably the best decision I have ever made. I look forward to the next journey with you (and Dylan). i Abstract The oxygenated volatile organic compound, acetaldehyde, plays a fundamental role in atmospheric chemistry by altering the atmosphere’s oxidative status or “self-cleaning capacity”. Acetaldehyde flux from freshwater, estuarine, and marine environments is thought to be under significant microbial control, yet the identity and diversity of microbial acetaldehyde degraders, and the mechanisms of acetaldehyde degradation, are largely unknown. In this thesis, the key acetaldehyde-degrading microorganisms in the Colne Estuary, the River Colne and the River Gipping were identified using metagenetic sequencing and quantitative PCR. Bacteria were primarily responsible for acetaldehyde degradation, with members of the genera Pseudomonas and Arcobacter identified as key acetaldehyde degraders. Seventeen bacterial isolates were cultivated from the Colne Estuary and degraded acetaldehyde at a concentration of 2.27 mM, with Halobacillus strains A4 and A5, Rhodococcus strain A14, and Bacillus strain A17 identified as important acetaldehyde degraders. Fourteen previously identified bacteria, belonging to the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia, also degraded acetaldehyde, suggesting that the microbial acetaldehyde sink is highly diverse. Using genomics and proteomics, the pathway of acetaldehyde metabolism used by Rhodococcus strain A14 was proposed. Acetaldehyde is oxidised to acetate by aldehyde dehydrogenase enzymes, with acetate converted to acetyl-CoA via acetyl-CoA synthetase. These reactions are thought to be conserved amongst acetaldehyde-degrading bacteria. Under favourable growth conditions, acetaldehyde is dissimilated to CO2 for energy production via the oxidative decarboxylation steps of the tricarboxylic acid cycle. Acetaldehyde-derived carbon is assimilated into biomass by upregulating the glyoxylate shunt and gluconeogenesis pathway, ensuring carbon is conserved for growth. Using 14C- radiolabelling, rates of microbial dissimilation were shown to be significantly higher than assimilation rates in the freshwater rivers, estuaries, and coastal seas of south-west ii England, with summer rates significantly higher than winter. These findings suggest that microorganisms exert considerable control over acetaldehyde emissions and represent an important component of the global acetaldehyde cycle. iii Contents Acknowledgements ................................................................................................................ i Abstract.................................................................................................................................. ii List of Tables ....................................................................................................................... vii Supplementary tables .......................................................................................................... vii List of Figures ..................................................................................................................... viii Supplementary figures .......................................................................................................... x Abbreviations ........................................................................................................................ xi Declaration ........................................................................................................................... xv Chapter 1: Introduction ................................................................................................... 1 1.1 Volatile organic compounds and the atmosphere ........................................................ 1 1.2 Acetaldehyde synthesis ............................................................................................... 5 1.3 Industrial and commercial significance......................................................................... 9 1.4 Health implications..................................................................................................... 12 1.5 Terrestrial sources and sinks ..................................................................................... 14 1.6 Marine sources .......................................................................................................... 17 1.7 Marine sinks .............................................................................................................. 23 1.8 Freshwater and estuarine sources and sinks ............................................................. 24 1.9 Aims and objectives ................................................................................................... 26 1.10 References .............................................................................................................. 30 Chapter 2: Microbial Degradation of Acetaldehyde in Freshwater and Estuarine Environments .................................................................................................................. 45 2.1 Introduction ................................................................................................................ 45 2.2 Methodology .............................................................................................................. 51 2.2.1 Sampling ............................................................................................................. 51 2.2.2 Enrichment preparation ....................................................................................... 53 2.2.3 Successive enrichments ..................................................................................... 56 2.2.4 Nutrient assimilation assays ................................................................................ 58 2.2.5 Microbial community analysis .............................................................................. 59 2.2.5.1 DNA extraction and quantitative-PCR ......................................................... 59 2.2.5.2 Metagenetic sequencing ............................................................................. 62 2.2.5.3 MiSeq bioinformatics ................................................................................... 65 2.2.6 14C-acetaldehyde enrichments ............................................................................ 65 2.2.7 Statistical analysis ............................................................................................... 66 2.3 Results ...................................................................................................................... 66 2.3.1 Acetaldehyde degradation and microbial abundance .......................................... 66 2.3.1.1 Colne Estuary microcosms .......................................................................... 66 2.3.1.2 River Colne and River Gipping microcosms ................................................ 74 2.3.2 Microbial community analysis .............................................................................. 77 2.3.2.1 Microbial communities of the Colne Estuary ................................................ 77 iv 2.3.2.2 Microbial communities of freshwater rivers .................................................. 91 2.3.3 Microbial 14C-acetaldehyde uptake .................................................................... 103 2.4 Discussion ............................................................................................................... 103 2.4.1 Microbial acetaldehyde degradation .................................................................. 103 2.4.2 Features of Pseudomonas species that facilitate acetaldehyde degradation ..... 109 2.4.3 Physiology