THE MICROBIOLOGY OF ACTIVATED SLUDGE

THE MICROBIOLOGY OF ACTIVATED SLUDGE

THE MICROBIOLOGY OF ACTIVATED SLUDGE Edited by R.J. SEVIOUR Biotechnology Research Centre La Trobe University, Bendigo, Victoria, 3550 Australia and L.L. BLACKALL Department of Microbiology, University of Queensland, Brisbane, Queensland, 4072 Australia SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A c.i.p. Catalogue record for this book is availab1e from the Library of Congress. ISBN 978-94-010-5754-7 ISBN 978-94-011-3951-9 (ebook) DOI 10.1007/978-94-011-3951-9 Printed an acid-free paper AH Rights Reserved 1999 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1999 Softcover reprint ofthe hardcover 1 st edition 1999 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, inc1uding photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

Contents Preface List of Contributors lx xi 1 Introduction to the microorganisms found in activated sludge processes R.J. Seviour and L.L. Blackall 1.1 Introduction 1 1.2 General features and characteristics of microbes relevant to activated sludge systems 5 1.3 What is microbiology? 6 1.4 How do we study microbes? 6 1.5 What are these microbes? 6 1.6 Microbial energetics 21 1.7 The growth and nutrition of microbes 34 1.8 Control and prevention of microbial growth 43 1.9 Conclusions 43 1 2 The activated sludge process 44 R.J. Seviour, K.c. Lindrea, P.c. Griffiths and L.L. Blackall 2.1 Introduction 2.2 Design configurations for activated sludge systems 2.3 Monitoring the process 2.4 Modelling the activated sludge process 2.5 The future of modelling activated sludge 44 46 62 68 74 3 The normal microbial communities of activated sludge plants R.J. Seviour 3.1 Introduction 3.2 The floc 3.3 Factors affecting survival of an organism in activated sludge systems 3.4 Methods for studying microbial populations in activated sludge 3.5 Microbial community composition 3.6 Manipulations of the microbial community in activated sludge plants 3.7 Conclusions 76 76 77 78 80 83 98 98

vi Contents 4 Factors affecting the occurrence of filamentous bacteria in activated sludge plants 99 R.J. Seviour 4.1 Introduction 99 4.2 Reasons for inadequate solids separation in clarifiers 99 4.3 Which factors determine the filamentous populations in different plants? 105 4.4 Strategies for filament control and modelling their growth 121 5 Current taxonomic status of filamentous bacteria found in activated sludge plants 122 R.J. Seviour and L.L. Blackall 5.1 Introduction 122 5.2 Characters used in the classification and identification of bacteria 125 5.3 How do we classify and identify the filamentous bacteria? 127 5.4 Current classifications of the filamentous bacteria 135 5.5 Current status of filament identification procedures 139 5.6 Future prospects for filament taxonomy 142 6 Bulking 147 1.L. Blackall 6.1 Introduction 147 6.2 The occurrence of bulking in activated sludge systems 147 6.3 The filaments responsible for bulking 150 6.4 Kinetic competition studies on cultures of bulking filamentous bacteria 153 6.5 Some theories to explain bulking 155 6.6 Control of bulking 158 6.7 Summary 159 7 Foaming 161 J. Soddell 7.1 Introduction 161 7.2 Which microbes cause foam? 162 7.3 Taxonomy of foam formers 172 7.4 How is foam formed? 182 7.5 Factors affecting growth of foam formers 186 7.6 Hydrophobic substrates 189 7.7 How do foam formers compete? 194 7.8 Control of foaming 195 7.9 Conclusions 201

Contents vii 8 The microbiology of nitrogen removal in activated sludge systems L.L. Blackall and P. Burrell 8.1 Introduction 8.2 The microbiology of nitrogen removal 8.3 The autotrophic ammonia-oxidizing or nitroso bacteria 8.4 Autotrophic nitrite-oxidizing or nitro bacteria 8.5 Nitrification in activated sludge 8.6 Denitrification 203 203 205 208 214 219 222 9 Microbiological aspects of phosphorus removal in activated sludge systems P.1. Bond and GN. Rees 9.1 Introduction 9.2 The process of EBPR 9.3 Development of biochemical models for phosphorus removal 9.4 Evaluation of bacterial populations in EBPR activated sludge 9.5 The taxonomic status of the genus Acinetobacter and its relevance to EBPR 9.6 Is the behaviour of pure cultures in accordance with the models? 9.7 Filamentous bacteria in EBPR plants 9.8 Bacterial inhibition of EBPR 9.9 Inoculation of activated sludge with polyphosphateaccumulating bacteria 9.10 Biochemical events associated with EBPR 9.11 Do polyphosphate transformations account for the phosphorus flux? 9.12 Localization and structure of bacterial polyphosphate 9.13 Bacterial metabolism of polyphosphate 9.14 Bacterial phosphate transport 9.15 Possible functions of poly P in activated sludge 9.16 The structure and metabolism of PHA in activated sludge 9.17 Bacterial glycogen metabolism 9.18 The imperative nature of the anaerobic zone 9.19 Conclusions 227 227 228 230 232 236 237 239 240 241 242 242 244 245 248 249 251 253 254 256 10 Practical methods for the examination and characterization of activated sludge 257 K.c. Lindrea, E.M. Seviour, KJ. Seviour, L.L. Blackall and J.A. Soddell 10.1 Introduction 257 10.2 The light microscope 257 10.3 Measurement of dimensions of microbial cells 264 10.4 The electron microscope 265 10.5 The confocal laser microscope 265 10.6 Preparation of specimens for microscopy 266

viii Contents 10.7 Stains used for examination of activated sludge samples 269 10.8 Settlability tests and qualitative estimation of filamentous populations in mixed liquor and foam samples 279 10.9 Filament estimation techniques 281 10.10 Determination of surface hydrophobicity of solids in foaming activated sludge plants 284 10.11 Isolation of filamentous bacteria from activated sludge 284 10.12 Use of gene probes for the in situ identification of filamentous bacteria in activated sludge samples 285 10.13 Analysis of biomass microfauna to determine the sludge biotic index (SBI) 291 10.14 Collection of data from microscopic analysis and use of worksheets 293 11 Descriptions of the filamentous bacteria causing bulking and foaming in activated sludge plants E.M. Seviour, R.J. Seviour and K.c. Lindrea 301 11.1 Identification of filamentous bacteria from microscopic examination of foam and mixed liquor samples 301 11.2 Other filamentous bacteria in activated sludge plants 336 11.3 Important non-filamentous bacterial populations in activated sludge plants 341 11.4 Unidentified filamentous bacteria in activated sludge samples 345 11.5 Other organisms seen in activated sludge 347 Glossary References Index 349 354 410

Preface This book has been a long time in preparation. Initially it grew out of our frustrating attempts over the past ten years to identify the filamentous bacteria seen in large numbers in most activated sludge plants, and the realization that we know very little about them and the other microbial populations in these systems. Unfortunately this book does not provide many answers to the problems these filamentous bacteria can cause, but we hope it might encourage microbiologists and engineers to communicate more with each other and to spend some time trying to understand the taxonomy, ecology and physiology of activated sludge microbes. It is now very timely, for example, to try to provide these filamentous bacteria with proper taxonomically valid names and to determine their correct place in bacterial classifications. This book is not meant to compete directly with the books by Gray (1989, 1990) nor the excellent manual published by Jenkins and coworkers (1993b), which has been invaluable to us and others trying to identify filamentous bacteria. Wanner's book (1994a) also provides an excellent account of the problems of bulking and foaming caused by filamentous bacteria. These publications and others by Eikelboom's group have made an enormous contribution to the study of filamentous bacteria, and will continue to do so. Instead, our book has attempted to adopt a more general microbiological slant, concentrating less on the engineering aspects of the operational problems these filamentous bacteria cause and considering the microbiology of activated sludge in a much broader context. We see it complementing not competing with the others, and as such the book should be useful to microbiologists and engineers both in academe and industry. We also decided that now was an appropriate time to review what is known about the general microbiology of activated sludge, especially with the impact molecular biology is certain to have on the methods becoming available to study this complex ecosystem (Kempe et al., 1993; Akkermans et al., 1995; Pickup and Saunders, 1996; Hurst, 1997; Amann et al., 1998). The book is divided into several chapters. The first chapters consist of a number of essays which set out to assess critically the literature and our current understanding of activated sludge bacteria and what they might do. The chapters were written with an emphasis on what we believe are some areas in which research is desperately needed. These are then followed by a section describing the methodology particularly appropriate to the study of bacteria causing bulking and foaming. Lastly, we have included an identification section consisting of coloured photographs and individual descriptions for the filamentous and other bacteria commonly seen in activated sludge plants. We have also tried to introduce engineers and microbiologists to the jargon each uses in the hope that more effective communication might occur between them. For example, the recommended engineering notation of Wanner (1994a) and Grady et al. (1996) to describe the metabolic groupings of organisms, especially their meaning of the terms 'substrate' and 'energy source' is quite different to that adopted by most microbiologists who are confused. Such differences cannot help communication. This book would not have been published without the generous financial assistance provided by the Victorian Education Foundation and the Victorian Depart-

x Preface ment of Conservation and Natural Resources. We would like to express our special thanks to Mr Ian Kennedy and Mr Warren Wealands for their support and encouragement, without which this book would never have been written. We would also like to acknowledge staff at the wastewater treatment plants who have willingly sent samples to us, and all the engineers, chemists and microbiologists who, often for different reasons, have an interest in these filamentous bacteria, and who have encouraged us to complete this book. Grateful thanks are given to our colleagues who provided photographs and diagrams for this book, especially Prof. F. Lingens, Mr K. Lindrea and Dr M. Wagner. The invaluable contributions of Carol Williams, Geoff Knight, Bronwyn DeGrey, Dr Helen Stratton and Anne Keyte are also gratefully acknowledged, as are the excellent typing skills of Liz Howard, Michelle Warwick, Elaine Norris and Sue Scott. Finally we would like to thank Pam Lewis, Ian Paterson, Danielle Garlick and Dr John Anderson of La Trobe University Press for their endless support and encouragement during the early days of the project, and later, Andrew Stammer who had enough confidence in the value of this book to see it through to this final version.

List of contributors L.L. Blackall Department of Microbiology, The University of Queensland, Brisbane, Queensland 4072, Australia P.L. Bond Department of Microbiology, The University of Queensland, Brisbane, Queensland 4072, Australia P.c. Burrell Department of Microbiology, The University of Queensland, Brisbane, Queensland 4072, Australia P.c. Griffiths Montgomery Watson Pty Ltd, Brisbane, Queensland 4064, Australia K.c. Lindrea Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3550, Australia G.N. Rees Freshwater Ecology Research Centre, Albury, NSW 2640, Australia E.M. Seviour Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3550, Australia R.J. Seviour Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3550, Australia J.A. Soddell Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3550, Australia