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Molecular Profiling of Microbial Population Dynamics in Environmental Water

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Molecular Profiling of Microbial Population Dynamics in Environmental Water Molecular profiling of microbial population dynamics in environmental water K Jordaan 12419559 Thesis submitted in fulfillment of the degree Philosophiae Doctor in Environmental Sciences at the Potchefstroom Campus of the North-West University Supervisor: Prof CC Bezuidenhout May 2015 “The LORD is the everlasting God, the Creator of the ends of the earth. He will not grow tired or weary, and his understanding no one can fathom. He gives strength to the weary and increases the power of the weak. Even youths grow tired and weary, and young men stumble and fall; but those who hope in the LORD will renew their strength. They will soar on wings like eagles; they will run and not grow weary, they will walk and not be faint.” Isaiah 40:28–31 ii ACKNOWLEDGEMENTS Having concluded this research, words cannot adequitly describe my gratitude to the LORD my God. Throughout this project I was constantly aware of God’s provision of personal and material means, opportunities, and spiritual guidance. “Surely the arm of the Lord was not too short to save, nor his ear too dull to hear.” – Isaiah 59: 1. All glory and praise goes to the Creator of heaven and earth. I gratefully acknowledge the following persons: Prof. Carlos Bezuidenhout, for supervision of this research and his patients and support throughout this project. Dr. Leon van Rensburg, for his invaluable support of this research and being there during the tough times. Completing this project would not have been possible without him. Prof. Damase Khasa, for providing the most wonderful and memorable opportunity at Université Laval, Québec, Canada. I am forever grateful for this opportunity, his hospitality and kindness will not be forgotten. Dr. Andre Comeau, for his support with the bioinformatics analysis. I kindly thank him for his patience in answering my neverending list of questions, and his willingness to share his knowledge. Marie-Evé Beaulieu, for her kindness and assistance during my stay in Québec City. To my family, friends (especially Ina and Hermoine) and significant other – I thank you for your encouragement, support, patience, motivation, and love, without you I will be lost. I cherish you in my heart and I love you all dearly. Mom and Dad, thank you for always being there for me, and doing your best for your daughters. The morals you taught us in life cultured strong, dedicated, and independent children. May “The LORD iii bless you and keep you; make His face shine on you and be gracious to you; turn His face towards you and give you peace”. – Numbers 6: 23–26. iv ABSTRACT Increasing socio-economic growth and development of South Africa’s freshwater systems require continuous augmentation of water sources to meet the growing water requirements of communities and industries. Anthropogenic disturbances have caused the water quality of many freshwater systems to drastically deteriorate due to constant disposal of domestic, industrial, and agricultural waste into surface waters. Government agencies make use of biomonitoring programmes to effectively manage the countries’ freshwater resources. These programmes use a variety of biological indicators (e.g., macroinvertebrates, fish, diatoms and algal species) and physico-chemical variables to determine the state of the environment. However, attempts to use microbial community structures as bioindicators of anthropogenic perturbations are greatly neglected. This study used molecular techniques (PCR-DGGE and 454-pyrosequencing) and multivariate analysis to develop a robust monitoring technique to determine the impacts of environmental disturbances on bacterial community compositions in river systems in the North West Province. Significant contributions made by this project included the establishment of a bacterial diversity framework for South African freshwater systems that are impacted by a variety of anthropogenic activities (e.g., urban and informal settlements, agriculture and mining). Furthermore, case studies demonstrated the prevalence of specific taxa at polluted sites, as well as positive and negative associations between taxa and environmental variables and pollutants. Finally, biogeochemical cycles could be partially matched to bacterial community structures in river systems. The first part of the project included a pilot study that investigated bacterial structures in a segment of the Vaal River in response to environmental parameters using molecular techniques and multivariate analysis. The most important observations made during this study included the generation of a larger bacterial diversity dataset by pyrosequencing compared to PCR-DGGE. In addition, metagenomic and multivariate analyses provided clues about potential biogeochemical roles of different taxa. The second and third part of the project included two case studies that investigated bacterial communities in the Mooi River and Wonderfonteinspruit in response to environmental activities. Both these systems are impacted by a variety of external sources such as urban and informal settlements, agriculture, and mining. The results demonstrated that perturbations nearby the Mooi River and Wonderfonteinspruit caused the overall water quality to deteriorate which in v turn had a profound impact on bacterial community composition. Bacterial community structures at reference/control sites (Muiskraal and Turffontein dolomitic eye) had overall high species diversity (richness and evenness), whereas polluted sites showed lower species diversity and were dominated by the Beta- and Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. In addition, various potential pathogens (e.g. Eschirichia/Shigella, Legionella, Staphylococcus, Streptococcus etc.) were identified at impacted sites. Multivariate analysis suggested that bacterial communities and certain taxa (Malikia, Algoriphagus, Rhodobacter, Brevundimonas and Sphingopyxis) at polluted sites were mainly impacted by temperature, pH, nutrient levels, and heavy metals. Finally, the proportion of nitrogen and sulphur bacteria corresponded well with the nitrogen and sulphur levels measured in the Wonderfonteinspruit. Based on these results, it was concluded that bacterial community structures might provide a good indicator of anthropogenic disturbances in freshwater systems and may be incorporated into biomonitoring programs. Keywords: freshwater; physico-chemical parameters; bacterial community composition; PCR-DGGE; 454-pyrosequencing; multivariate analysis vi TABLE OF CONTENTS Acknowledgements----------------------------------------------------------------------------- iii Abstract--------------------------------------------------------------------------------------------- v List of Tables-------------------------------------------------------------------------------------- xi List of Figures------------------------------------------------------------------------------------- xiii CHAPTER 1: Introduction and Problem statement----------------------------------- 1 1.1 Microbial ecology in aquatic ecosystems------------------------------------- 1 1.2 Common bacterial lineages in freshwater systems------------------------ 2 1.2.1 Proteobacteria---------------------------------------------------------------- 2 1.2.2 Actinobacteria---------------------------------------------------------------- 3 1.2.3 Bacteroidetes----------------------------------------------------------------- 3 1.2.4 Cyanobacteria---------------------------------------------------------------- 3 1.2.5 Minor phyla-------------------------------------------------------------------- 4 1.3 Temporal and spatial variation in bacterial communities---------------- 5 1.3.1 Temporal variation---------------------------------------------------------- 6 1.3.2 Spatial variation-------------------------------------------------------------- 6 1.4 Microbial processes------------------------------------------------------------------- 8 1.4.1 Carbon cycle------------------------------------------------------------------ 8 1.4.2 Nitrogen cycle---------------------------------------------------------------- 9 1.4.3 Sulphur cycle----------------------------------------------------------------- 10 1.4.4 Phosphorus cycle----------------------------------------------------------- 12 1.5 Physico-chemical impacts on microbial community structures------- 13 1.5.1 Temperature, pH and salinity------------------------------------------- 13 1.5.2 Dissolved Organic Matter------------------------------------------------- 15 1.6 Anthropogenic impacts on bacterial community structures------------ 16 1.7 Microorganisms as bioindicators------------------------------------------------ 16 1.8 Molecular techniques----------------------------------------------------------------- 17 1.9 Community fingerprinting methods--------------------------------------------- 18 1.9.1 Denaturing Gradient Gel Electrophoresis (DGGE)---------------- 18 1.10 Metagenomics-------------------------------------------------------------------------- 20 1.11 Multivariate analysis of environmental data---------------------------------- 22 1.11.1 Principal component analysis (PCA)----------------------------------- 24 vii 1.11.2 Non-metric multidimensional scaling (NMDS)----------------------- 24 1.11.3 Redundancy analysis (RDA)--------------------------------------------- 25 1.11.4 Canonical correspondence analysis (CCA)--------------------------- 25 1.12 Problem statement-------------------------------------------------------------------- 25 1.13 Outline of the thesis-------------------------------------------------------------------
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