Bacteria-Protozoan Interactions in a Mixed-Species Biofilm Community
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Bacteria-Protozoan Interactions in a Mixed- Species Biofilm Community Lee Sia Tan A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia August, 2010 Abstract Environmental biofilms typically consist of mixed-species communities exhibiting complex inter- and intra-species synergistic/antagonistic interactions. Intense competition in aquatic systems is driven by nutrient and space limitation. Bacterial biofilm members cooperate under such conditions to resist environmental perturbations, processes that subsequently determine community composition. This thesis reports on environmental bacterial screens for amensal/antagonistic metabolites against protozoa and nematodes. Further, defined mixed-species model communities isolated from the surface of the green alga Ulva australis were tested for their protozoan predation response under varying nutrient conditions. Biofilms exhibited higher levels of antiprotozoal and anthelmintic activity than their planktonic counterparts. Biofilm inhibitory activities were highly diverse, commonly targeting either nematodes or protozoa, but rarely targeting both with broad-spectrum activity. Predation resistance was explored using a model marine four-species community. Protozoan grazers of different feeding types were used on different-stage biofilms; Rhynchomonas nasuta (early-stage), Tetrahymena pyriformis and Acanthamoeba castellanii (intermediate- and late-stage). As single-species biofilms, all four species displayed varying levels of grazing. All were susceptible to R. nasuta whereas only Dokdonia donghaensis and Acinetobacter lwoffii were vulnerable to T. pyriformis. A. castellanii selectively preferred Shewanella japonica and A. lwoffii. Mixed-species biofilms facilitated synergistic and antagonistic interactions between consortia members. R. nasuta selectively preferred A. lwoffii, resulting in a ‘reverse grazer’ effect, allowing other members to benefit. T. pyriformis preferred grazing on D. donghaensis. The mixed-species community was resistant to A. castellanii grazing and thus commensal to D. donghaensis and A. lwoffii. Under high nutrient conditions, both three- and four-species biofilms resisted predation. By comparison, low nutrient conditions increased both mixed- and single-species biofilms susceptibility to A. castellanii grazing. Under low nutrient conditions, the removal of Microbacterium phyllosphaerae from the four-species consortium destabilized the community. Under low carbon conditions, S. japonica i demonstrated increased grazing susceptibility, possibly due to resource reallocation from defence to growth/maintenance. Bacterial grazing resistance depends on survival strategies, e.g. the ability to exploit nutrients and the trade-off between growth and defence. The differential levels of grazing resistance of both mixed- and single-species consortia, resulting from ‘top-down’ and ‘bottom-up’ factors, affected community structure and composition. Successful application of the carbon:nutrient balance hypothesis to S. japonica’s defence strategy, highlights the potential for eukaryotic theories as predictive models for microbial systems. ii List of Publications Tan, L. S., Matz, C., McDougald, D. and S. Kjelleberg. 2008. Impact of selective grazing by Acanthamoebae castellanii on a marine mixed-species biofilm. 12th International Symposium on Microbial Ecology Abstract Book. Tan, L. S., Matz, C., Steinberg, P., McDougald, D. M. and S. Kjelleberg. 2009. Effects of protozoan grazing on a mixed-species marine biofilm. ASM Conferences Biofilms 2009 Abstract Book. Tan, L. S., Steinberg, P., Matz, C., Kjelleberg, S. and D. McDougald. 2010. The effect of marine predators on the community composition in a marine mixed- species biofilm. 13th International Symposium on Microbial Ecology Abstract Book. iii Acknowledgements Firstly, I would like to thank my supervisor, Professor Staffan Kjelleberg for the opportunity he has given me to explore the microbial ecology of mixed species biofilms. He has provided me with wise and balanced guidance throughout my PhD candidature. Despite having a busy schedule and responsibilities, he never fails to ensure the well-being of his students. Most importantly, I am grateful to him for being my pillar of support though my toughest times at the last stage of my program, encouraging me to strive on. My co-supervisors have both been fantastic, especially Dr Diane McDougald, who was constantly there for me whenever I needed help, providing me with ideas in biofilm work, motivation and support. Her constructive suggestions and critical review of my report writing has been really helpful in piecing together the final chapters of this thesis. Dr Carsten Matz has also provided me with invaluable insights into the field of protozoology. His expertise in this area has taught me skills that have been beneficial in handling protists. I would also like to thank Professor Peter Steinberg, for introducing me to eukaryotic ecology, thus allowing me to expand my work into microbial ecology and community ecology. It has been exciting to apply such theories as predictive models into microbial systems. Dr Sharon Longford has been a gem, being willing to help at such last minute notice, providing valuable contributions to refining the thesis on a whole. Bill O’ Sullivan has been brilliant and provided expert help in proof reading this thesis. He has been patient at picking out and explaining the common mistakes and redundancies in this thesis. Many thanks to Adam Abdool and Kirsty Collard who have provided the Centre for Marine Bio-Innovation (CMB) with the most efficient administrative and logistic support. They have been very approachable when support was needed at various stages of the PhD candidature. Amy Pham, Charmaine Ng, Janice Hui, Jerry Woo and Lan Le have been wonderful friends, being both supportive and encouraging through times when PhD seems really tough. iv I would also like to thank my parents for being such great models, both of whom have provided me with endless support and encouragement, for providing me with advice on dealing with life’s unexpected events. Last but not least, the University of New South Wales, Graduate Research School (GRS) and the Environmental Biotechnology CRC (EBCRC) have been generous at providing scholarship and travel fundings during my PhD candidature for various aspects such as extension of my PhD and financing my attendance for 2 international conferences in Cairns and Mexico. The School of Biotechnology and Biomolecular Sciences (BABS) has also been supportive in financing my 6 month extension, which I am grateful for. v Table of Contents Abstract .................................................................................................................... i List of Publications ................................................................................................ iii Acknowledgements ................................................................................................ iv Table of Contents ................................................................................................... vi List of Figures ...................................................................................................... xiii List of Tables .............................................................................................. xxi Abbreviations ...................................................................................................... xxii Chapter 1: Literature Review .................................................................................. 1 1.1 Introduction ................................................................................................... 1 1.2 Marine ecosystems ........................................................................................ 2 1.3 The biofilm mode of life ............................................................................... 3 1.3.1 The biofilm structure ............................................................................ 4 1.3.2 Biofilm sociobiology ............................................................................ 5 1.3.2.1 Interactions within mono-species biofilms ........................................ 6 1.3.2.2 Interactive behavior of mixed-species biofilms ................................. 7 1.3.3 Biofilms as a reservoir of bioactives .................................................. 10 1.4 Protozoa ...................................................................................................... 12 1.4.1 General classification of protozoa ...................................................... 12 1.4.2 Protozoan metabolism ........................................................................ 17 1.5 Predator-prey interactions ........................................................................... 18 1.5.1 Protozoan feeding preferences ............................................................ 18 1.5.2 Ecological impacts of grazing on the marine ecosystem and its microbial communities ................................................................................ 21 vi 1.5.3 Application of eukaryotic ecological theory as a conceptual framework for microbial ecology ................................................................................... 24 1.5.4 Grazing resistance mechanisms .........................................................