İSTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY MICROBIAL COMMUNITY AND METABOLISM OF ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL Ph.D. Thesis by Gülsüm Emel ZENGİN Department : Environmental Engineering Programme : Environmental Engineering JANUARY 2009 İSTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY MICROBIAL COMMUNITY AND METABOLISM OF ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL Ph.D. Thesis by Gülsüm Emel ZENGİN, M.Sc. (501002355) Date of submission : 15 September 2008 Date of defence examination: 13 January 2009 Supervisor (Chairman) : Prof. Dr. Nazik ARTAN (ITU) Co-supervisor : Prof. Dr. Takashi MINO (UT) Members of the Examining Committee : Prof. Dr. Derin ORHON (ITU) Prof. Dr. Orhan YENİGÜN (BU) Prof. Dr. Rüya TAŞLI TORAMAN(ITU) Prof. Dr. Delya SPONZA (DEU) JANUARY 2009 İSTANBUL TEKNİK ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİK AŞIRI FOSFOR GİDERİMİNİN MİKROBİYAL TÜRLERİ VE METABOLİZMASI DOKTORA TEZİ Y. Müh. Gülsüm Emel ZENGİN (501002355) Tezin Enstitüye Verildiği Tarih : 15 Eylül 2008 Tezin Savunulduğu Tarih : 13 Ocak 2009 Tez Danışmanı : Prof. Dr. Nazik ARTAN (İTÜ) Eş Danışman : Prof. Dr. Takashi MINO (UT) Diğer Jüri Üyeleri : Prof. Dr. Derin ORHON (İTÜ) Prof. Dr. Orhan YENİGÜN (BÜ) Prof. Dr. Rüya TAŞLI TORAMAN (İTÜ) Prof. Dr. Delya SPONZA (DEÜ) OCAK 2009 ACKNOWLEDGMENT I would like to express my sincere gratitude and appreciation to my supervisors Prof. Dr. Nazik Artan and Prof. Dr. Takashi Mino for their effort in providing me their experience and knowledge, for the encouragement and support throughout my study and especially for their understanding. I would especially like to thank Prof. Dr. Derin Orhon for his sincere support, inputs for my study, and specially his encouragement and help for giving opportunity to study abroad. I would like to express my deepest gratitude to Assoc. Prof. Dr. Hiroyasu Satoh for his sincere help and very valuable contributions during my study in laboratory and extending his experience and knowledge during evaluating the results. I would like thank my committee member, Prof. Dr. Orhan Yenigün, for his contributions to my PhD study. I would like to thank Japanese Government for providing me an opportunity with Monbukagakusho:MEXT 2003 Research Scholarship to study in the University of Tokyo where I carried out all my experimental work of my PhD study. I would like to thank all the academic staff and the techical staff of 14th and 9th building in the University of Tokyo for their support during my stay in Todai, specially, Dr. Onuki, Okamoto-san, Michinaka-san and all the members of Mino-ken. My deepest thanks to Adeline Seak Chua May, Pınar-Tuğser-Tuna Sarıünal for their truly friendship and their great support in Tokyo. I would like to acknowledge all my friends for their sincere support and kindness, specifically to Melike Gürel, Serdar Doğruel, Alpaslan Ekdal, Elif Pehlivanoğlu- Mantaş, Egemen Aydın, Nevin Yağcı and İlke Pala for their truly friendship and , great support throughout my study. I would like to thank Assoc. Prof. Dr. Emine Ubay Çokgör and Prof. Dr. Rüya Taşlı Toraman for many favors they have extended me. My beloved Levent, I am grateful for your love, patience, understanding and encouragement. Finally, my special thanks are to my dearest mother, father and brother, for their guidance and love they have afforded me. This thesis is dedicated to my father for his belief in me and for the wisdom he has afforded me. September 2008 Gülsüm Emel ZENGİN iii TABLE OF CONTENTS Page NOMENCLATURE...................................................................................................vii LIST OF TABLES .....................................................................................................ix LIST OF FIGURES....................................................................................................xi ÖZET .......................................................................................................................xv SUMMARY.............................................................................................................xvii 1. INTRODUCTION....................................................................................................1 2. LITERATURE REVIEW..........................................................................................5 2.1 Enhanced Biological Phosphorus Removal (EBPR) Mechanism .....................5 2.2 Enhanced Biological Phosphorus Removal with Amino Acids .........................9 2.2.1 Fermentation of amino acid .....................................................................10 2.2.1.1 Fermentation of aspartate..............................................................15 2.2.1.2 Fermentation of glutamate.............................................................20 2.2.2 Metabolism of fermentation end products by phosphate accumulating organisms ...............................................................................................26 2.3 Enhanced Biological Phosphorus Removal with Glucose ..............................31 2.4 Microbiology of Enhanced Biological Phosphorus Removal...........................34 3. MATERIALS AND METHODS.............................................................................39 3.1 Laboratory Scale Sequencing Batch Reactors (SBRs) ..................................39 3.2 Activated Sludge Sample................................................................................40 3.3 Synthetic Wastewater .....................................................................................41 3.4 Analyses .........................................................................................................42 3.4.1 Ammonium nitrogen ................................................................................43 3.4.2 Anions......................................................................................................44 3.4.3 Volatile fatty acids....................................................................................44 3.4.4 Dissolved organic carbon ........................................................................44 3.4.5 Glycogen .................................................................................................44 3.4.6 Mixed liquor suspended solids and volatile suspended solids ................45 3.4.7 Total phosphorus.....................................................................................45 3.4.8 Polyhydroxyalkaonate .............................................................................46 3.4.9 Amino acids .............................................................................................46 3.4.10 Sampling and DNA extraction ...............................................................48 3.4.11 Polymerase chain reaction (PCR) .........................................................48 3.4.12 Agarose gel check .................................................................................48 3.4.13 Denaturing gradient gel electropheresis and sequencing .....................49 3.4.14 Cloning and sequencing ........................................................................50 3.4.15 Probe design .........................................................................................51 3.4.16 FISH ......................................................................................................51 3.4.17 Microscopic analysis..............................................................................53 4.RESULTS AND DISCUSSION..............................................................................55 4.1 Enhanced Biological Phosphorus Removal in a Sequencing Batch Reactor (SBR) with Glucose .........................................................................................55 4.1.1 SBR-1 performance.................................................................................56 4.1.2 Microbial analyses of SBR-1 activated sludge ........................................62 4.1.3 Discussion ...............................................................................................77 v 4.2 Enhanced Biological Phosphorus Removal in a Sequencing Batch Reactor Aspartate and Glutamate ..................................................................................80 4.2.1 EBPR performance with aspartate and glutamate in SBR-2 and batch tests ........................................................................................................81 4.2.2 Microbial analyses of SBR-2 activated sludge ........................................89 4.2.3 Anaerobic stoichiometry of aspartate and glutamate in enhanced biological phosphorus removal .............................................................106 4.2.3.1 Anaerobic stoichiometry of aspartate ..........................................109 4.2.3.2 Anaerobic stoichiometry of glutamate .........................................125 4.2.4 Discussion...............................................................................................130 5. CONCLUSIONS.................................................................................................135 REFERENCES.......................................................................................................141 APPENDICES ........................................................................................................151 CURRICULUM VITAE ...........................................................................................193 vi NOMENCLATURE 3H2MB : 3-Hydroxy-2-Methylbutyrate 3H2MV : 3-Hydroxy-2-Methylvalerate