MECHANISMS OF DIVERSITY MAINTENANCE, RESILIENCE, AND NICHE DIFFERENTIATION IN ACTIVATED SLUDGE MICROBIAL COMMUNITIES By David Charles Vuono A thesis submitted to the Faculty and Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Environmental Science and Engineering). Golden, Colorado Date __________________ Signed:___________________ David C. Vuono Signed:___________________ Dr. Jörg E. Drewes Thesis Advisor Signed:___________________ Dr. Junko Munakata Marr Thesis Co-Advisor Golden, Colorado Date __________________ Signed:___________________ Dr. John E. McCray Professor and Department Head Department of Civil and Environmental Engineering ii 1 ABSTRACT Scarcity of potable water is a major threat to human civilization, particularly in semi-arid and arid regions of the world. As a consequence, water reclamation and reuse of treated wastewater has received more attention. Local reuse of reclaimed water that is tailored to local needs will be essential in cities because of greater population densities. Because it has been applied under many operational configurations, the activated sludge process was chosen to investigate if new operational strategies could facilitate tailored reuse of reclaimed water. However, alterations of operational conditions within the same biological nutrient removal treatment plant have not been tested for the intentional retention of nutrients in treated wastewater. Thus, the consequences of changing treatment conditions on biological and engineered operational parameters in a treatment plant are unknown, specifically the resilience of functionally important microorganisms involved in the removal of nutrients or trace organic chemicals. This dissertation describes the use of a variety of genomic molecular techniques such as massively parallel DNA/RNA sequencing to phylogenetically reconstruct activated sludge microbial community assemblages in a full-scale active sludge wastewater treatment bioreactor that is configured as a sequencing batch membrane bioreactor (SBMBR). The SBMBR system, located at the Colorado School of Mines’ Water Reclamation Test Site, was operated under different bioreactor set-points through time to investigate fundamental and applied questions in the fields of microbial ecology and environmental engineering. The key findings of each research chapter are as follows: Chapter 2) Adjustments in aeration strategy can be used to elevate nutrient concentrations in the effluent of an SBMBR without adverse effects on bioreactor operation. Chapter 3) Bioreactor performance in an SBMBR is resilient to long-term disturbances. Abundant bacterial populations recover from the disturbance more than rare populations. Chapter 4) A long-term disturbance allows for immigrating bacteria to colonize and establish into an activated sludge community. In some cases, newly established colonizers become abundant community members post disturbance. Chapter 5) Bacterial populations within a stable treatment process exhibit distinct activity signatures that correspond to fluctuations in redox conditions. iii 2 TABLE OF CONTENTS 1 ABSTRACT ............................................................................................................................. iii 3 LIST OF FIGURES ............................................................................................................... viii 4 LIST OF TABLES .................................................................................................................... x 5 ACKNOWLEDGEMENTS ..................................................................................................... xi 6 DEDICATION ........................................................................................................................ xii 1 CHAPTER 1 ............................................................................................................................. 1 1.1 Background ................................................................................................................... 1 1.2 Research Motivation ..................................................................................................... 3 1.3 Research Objectives ...................................................................................................... 4 2 CHAPTER 2 – ENGINEERING AND OPERATIONAL CHALLENGES FOR FLEXIBLE HYBRID TREATMENT SYSTEMS ............................................................................................. 6 2.1 Abstract ......................................................................................................................... 6 2.2 Introduction ................................................................................................................... 6 2.3 Materials and Methods .................................................................................................. 8 2.3.1 Site layout and SBMBR process description .................................................... 9 2.3.2 Description of treatment strategies. ................................................................ 11 2.3.3 Data acquisition and analytical procedures ..................................................... 11 2.3.4 Statistical Analysis .......................................................................................... 13 2.4 Results and Discussion ............................................................................................... 13 2.4.1 Start up ............................................................................................................ 13 2.4.2 Treatment Performance under Strategies 1 and 2 ........................................... 14 2.4.3 Membrane performance .................................................................................. 17 2.4.4 Conclusion ...................................................................................................... 21 2.5 Acknowledgments ....................................................................................................... 21 3 CHAPTER 3 – PHYLOGENETIC RECONSTRUCTION OF AN ACTIVATED SLUDGE COMMUNITY UNDER DIFFERENT SOLIDS RETENTION TIMES ..................................... 22 3.1 Abstract ....................................................................................................................... 22 3.2 Introduction ................................................................................................................. 23 3.3 Materials and Methods ................................................................................................ 25 iv 3.3.1 Sampling, Amplification, and Sequencing ..................................................... 25 3.3.2 SSU rRNA processing pipeline and quality control ....................................... 25 3.4 Results ......................................................................................................................... 26 3.4.1 Analysis of Ecosystem Function ..................................................................... 26 3.4.2 Effects of SRT on Bacterial Diversity ............................................................ 27 3.4.3 Effects of SRT on Community Composition .................................................. 30 3.4.4 Bacterial Dynamics/Response During Selective Enrichment of r-K Strategists 31 3.5 Discussion ................................................................................................................... 34 3.5.1 Microbial Diversity is Not Resilient to All Types of Disturbance ................. 34 3.5.2 Improved Methods of Comparing Microbial Diversity .................................. 36 3.5.3 Prediction of Ecosystem Health Based on Indicator Species ......................... 36 3.5.4 Merging Ecology and Environmental Engineering ........................................ 37 3.6 Acknowledgments ....................................................................................................... 37 4 CHAPTER 4 – IMMIGRATION AND MECHANISMS OF MICROBIAL DIVERSITY MAINTENANCE IN ACTIVATED SLUDGE ........................................................................... 37 4.1 Abstract ....................................................................................................................... 38 4.2 Introduction ................................................................................................................. 38 4.3 Materials and Methods ................................................................................................ 40 4.3.1 Bioreactor operation ........................................................................................ 40 4.3.2 Sampling, Amplification, and Sequencing ..................................................... 40 4.3.3 SSU rRNA processing and quality control ..................................................... 41 4.3.4 Biodiversity analysis, null model consideration and network analysis .......... 41 4.4 Results ......................................................................................................................... 43 4.4.1 Bacterial composition and diversity of sewage-derived and sludge communities ................................................................................................................ 44 4.4.2 Disturbance drives phylogenetic community structure ................................... 45 4.4.3 Activated sludge communities become more similar to influent during disturbance .................................................................................................................