Over the past twenty years, the knowledge and understanding of wastewater treatment has advanced extensively and moved away from empirically based approaches to a fundamentally-based first principles approach embracing chemistry, microbiology, and physical and bioprocess engineering, often involving experimental laboratory work and techniques. Many of these experimental methods and techniques have matured to the degree that they have been accepted as reliable tools in wastewater treatment research and practice. For sector professionals, especially a new generation of young scientists and engineers entering the wastewater treatment profession, the quantity, complexity and diversity of these new developments can be overwhelming, particularly in developing countries where access to advanced level laboratory courses in wastewater treatment is not readily available. In addition, information on innovative experimental methods is scattered across scientific literature and only partially available in the form of textbooks or guidelines. This book seeks to address these deficiencies. It assembles and integrates the innovative experimental methods developed by research groups and practitioners around the world. Experimental Methods in Wastewater Treatment forms part of the internet-based curriculum in wastewater treatment at UNESCO-IHE and, as such, may also be used together with video records of experimental methods performed and narrated by the authors including guidelines on what to do and what not to do. The book is written for undergraduate and postgraduate students, researchers, laboratory staff, plant operators, consultants, and other sector professionals.

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Experimental Methods in Wastewater Treatment

By Mark C. M. van Loosdrecht, Per H. Nielsen, Carlos M. Lopez-Vazquez, Damir Brdjanovic

IWA Publishing

1. INTRODUCTION Mark C.M. van Loosdrecht, Per H. Nielsen, Carlos M. Lopez-Vazquez and Damir Brdjanovic (aut.), 1, 
2. ACTIVATED SLUDGE ACTIVITY TESTS Carlos M. Lopez-Vazquez, Laurens Welles, Tommaso Lotti, Elena Ficara, Eldon R. Rene, Tessa P.H. van den Brand, Damir Brdjanovic and Mark C.M. van Loosdrecht (aut.) Yves Comeau, Piet N.L. Lens and Nancy G. Love (rev.), 7, 
3. RESPIROMETRY Henry Spanjers and Peter A. Vanrolleghem (aut.) George A. Ekama and M. Sperandio (rev.), 133, 
4. OFF-GAS EMISSION TESTS Kartik Chandran, Eveline I.P. Volcke, Mark C.M. van Loosdrecht (aut.) Peter A. Vanrollegem and Sylvie Guillot (rev.), 177, 
5. DATA HANDLING AND PARAMETER ESTIMATION Gurkan Sin and Krist V. Gernaey (aut.) Sebastiaan C.F. Meijer and Juan A. Baeza (rev.), 201, 
6. SETTLING TESTS Elena Torfs, Ingmar Nopens, Mari K.H. Winkler, Peter A. Vanrolleghem, Sophie Balemans and Ilse Y. Smets (aut.) Glenn T. Daigger and Imre Takacs (rev.), 235, 
7. MICROSCOPY Jeppe L. Nielsen, Robert J. Seviour and Per H. Nielsen (aut.) Jin Wanner (rev.), 263, 
8. MOLECULAR METHODS S�ren M. Karst, Mads Albertsen, Rasmus H. Kirkegaard, Morten S. Dueholm and Per H. Nielsen (aut.) Holger Daims (rev.), 285, 
LIST OF SYMBOLS AND ABBREVIATIONS,

CHAPTER 1

INTRODUCTION

Authors: Mark C.M. van Loosdrecht Per H. Nielsen Carlos M. Lopez-Vazquez Damir Brdjanovic

Wastewater treatment forms a crucial link in the services that the sanitation sector delivers to society. For centuries, sanitation largely consisted of transporting fresh, clean water to the cities, and using this water to transport the waste out of the city and discharge it into the natural environment. However, with the increase in human populations in cities as a result of the industrial revolution in the 19 century, this could no longer be maintained. The occurrence of epidemic diseases facilitated the development of wastewater treatment facilities and their implementation since the early 20th century. This development has been largely an empirical activity with theoretical approaches following experimental observations (Figure 1.1).

The discovery and development of activated sludge technology (described in detail in Jenkins and Wanner, 2014) was crucial as it triggered the rapid development and application of various analytical and experimental methods. Experimental work in the Lawrence Experimental Station in Massachusetts, USA, which at that time (1912) was a unique facility aimed at the experimental verification of different possible wastewater treatment procedures, inspired Gilbert Fowler to request Edward Ardern and William Lockett to repeat the experiments with wastewater aeration in the UK that he had seen in the USA. In 1913 and 1914 Lockett and Ardern carried out lab-scale experiments at the Manchester - Davyhulme wastewater treatment plant (Figure 1.2). Glass bottles were used to represent labscale aeration basins 'fed' by sewage from different districts of Manchester. Contrary to the experiments that Fowler saw in Massachusetts, in the Manchester aeration tests the sediment that remained after decantation was left in the bottle and a new dose of sewage was added to the sediment for the next batch. Lockett and Ardern soon found that the amount of the sediment increased with the increasing number of batches. At the same time the aeration time necessary for 'full oxidation' of sewage (full oxidation was a term used to describe the removal of degradable organics and for complete nitrification) was reduced. By using this technique of repeated batch aeration with the sediment remaining in the bottle, Lockett and Ardern were able to shorten the required aeration time for 'full oxidation' from a few weeks to less than one day, which made the process technically feasible. The sediment formed during the aeration of sewage was called activated sludge due to its appearance and activity. Lockett and Ardern published their results in a famous series of three papers (Ardern and Lockett 1914a, 1914b, 1915). This was the 'birth' of activated sludge, which is today the workhorse of wastewater treatment and the most widely applied sewage treatment technology in the world.

Wastewater engineering is a profession that is extremely experiment-based, and therefore it has always had the need to develop and standardise methods. This seemingly simple activity is strongly hampered by two factors, namely: (i) wastewater engineering is a typical interdisciplinary activity where chemical engineers, civil engineers, microbiologists and chemists interact to develop and understand the processes; the challenge here is to integrate methods and approaches from these disciplines, and, (ii) in addition, wastewater and its treatment processes are by their nature difficult to define with exactitude. It is for instance virtually impossible to measure all the individual compounds in the wastewater itself. Identifying all the relevant microorganisms in the processes has long been impossible and is still a complicated challenge. Defining all the potentially occurring chemical conversions is, due to the myriad of chemicals present, again an almost impossible task.

Due to the undefined nature of the experimental system, research has tended to progress slowly and it heavily depends on standardised methods that may not be exact but, when used in a standardised way, are very helpful and useful to compare experimental results. Examples are the commonly used chemical or biological oxygen demand tests. The iconic 'Standard Methods for the Examination of Water and Wastewater' (APHA et al., 2012, Figure 1.3) has for generations of sanitary engineers been the resource for analysing their experimental systems and full-scale operations. These methods focus heavily on the chemical characterization and measurement of specific microorganisms.

Societal demands on the efficiency of wastewater treatment plants have advanced, moving from public health protection to water resources and environmental protection and nowadays to integrated resource and energy recovery. Therefore the need to accurately characterize the microbial processes in the wastewater treatment processes has increased over recent decades. Certainly, it is a challenge to develop standardized methods for experimental work that can be easily repeated in different laboratories. In many cases, the exact handling is important, but it is not easy to be written down in a practical protocol.

Therefore, to avoid these problems, it was decided to develop not only a book describing all the experimental methods but also a video catalogue with the methods described in this book actually being demonstrated in the laboratory. This book and its associated video-based material are designed to support the research and development field with a manual for characterizing the biological processes in wastewater treatment. The editors have decided in this first edition of the 'Experimental methods in wastewater treatment' book to focus on the activated sludge process since this is worldwide by far the most applied technology. Nevertheless, most of the methods presented in this book can also be applicable to biofilm-based technologies or anaerobic digestion processes.

The decision to focus on experimental methods related to the activated sludge process has resulted in seven chapters describing the key experimental methods. The content and focus of these chapters are summarised in Table 1.1. Activated sludge consists of a myriad of microorganisms, converting a range of important compounds (organic matter, oxygen, nitrogen and phosphate compounds). The first three chapters focus on characterizing the conversion capacities of the microbial communities for the major microbial processes. A distinction has been made between full liquid-phase-based methods and methods where the conversion are characterized by measuring the respiration of the organisms, usually gas-phase measurements. Since there is an increasing focus on and interest in assessing the environmental impact of wastewater treatment plants, a separate chapter has been added for measuring greenhouse gas emissions from wastewater treatment plants. These chapters are followed by a chapter describing data handling techniques. Measurements often, certainly from full-scale or pilot plants, have relatively large uncertainties. With adequate data handling techniques the measurements can be used to derive associated (difficult to directly measure) process data or to minimise their uncertainty.

Activated sludge processes mainly depend on settling of the flocculent sludge to separate the biomass from the cleaned wastewater. This is often the Achilles heel of the treatment process and a key factor in the process design. One chapter is therefore devoted to characterization of the sludge settling properties.

As said earlier, microorganisms are the workhorses in the activated sludge process. Therefore the microscope is unavoidably the main technique to observe them directly, not only for individual organisms but also for the floc morphology related to settling characteristics. For a long time the microscope has been the main method of choice when observing which bacteria are present in activated sludge. However, although very helpful, it cannot show the full complexity of the microbial community. The last decade's advance in molecular DNA-based techniques has revolutionized the way one can observe microorganisms. These generic novel methods are described in the final chapter of this book.

Within the chapters the authors have tried to describe especially those methods that are experimentally complex and not standard analytical procedures. Therefore, standard analytical methods for e.g. organic matter, ammonium, phosphate etc. are not described in detail. On the other hand, it was also decided to include some analytical techniques recently developed and/or improved that are becoming frequently used but are scattered across scientific literature (e.g. glycogen and poly-hydroxy-alkanoates determination). In addition, methods that could be of academic interest but currently have limited practical application have not been included in detail in the text.

In terms of symbols and notation, an attempt has been made to standardize them as much as possible. While this was achieved at the chapter level, full standardization was not possible across all the chapters due to their diverse nature and heterogeneity of items as well as lack of global agreement on the use of symbols and notations, although the most common guidelines were quite closely followed (e.g. Corominas et al., 2010).

The book is conceptualized so as to satisfy users with high demands who are able to handle complex analytical and experimental equipment. However, the content is equally suited to the requirements of less advanced laboratories and less experienced experimenters; in particular, the complementary, freely available video materials address the execution of experiments in more challenging environments, such as those usually prevailing in most less developed countries.

"To measure is to know." Lord Kelvin

CHAPTER 2

ACTIVATED SLUDGE ACTIVITY TESTS

Authors: Carlos M. Lopez-Vazquez Laurens Welles Tommaso Lotti Elena Ficara Eldon R. Rene Tessa P.H. van den Brand Damir Brdjanovic Mark C.M. van Loosdrecht

Reviewers: Yves Comeau Piet N.L. Lens Nancy G. Love

INTRODUCTION

Different conditions and factors affect the degree and rate (speed) at which the compounds and contaminants of concern are removed by microbial populations in biological wastewater treatment systems. Certainly, the plant configuration and operational conditions play a major role in the prevalence of specific microbial populations and their activities, but factors as diverse and broad as wastewater characteristics and environmental and climate conditions have a strong influence as well. Eventually, in any biological wastewater treatment system, there will be a need to assess, define and understand the plant performance with regard to the removal of certain contaminants and the response of the sludge to inhibitory or toxic compounds of interest. Moreover, from a modelling perspective it is also of interest to assess and determine the stoichiometry and kinetic rates of the conversion processes performed by specific microbial populations (e.g. ordinary heterotrophic organisms: OHOs; denitrifying ordinary heterotrophic organisms: dOHOs; ammonium-oxidizing organisms: AOOs; nitrite-oxidizing organisms: NOOs; phosphate-accumulating organisms: PAOs; sulphate-reducing bacteria: SRB, also identified as sulphate-reducing organisms, SRO (Corominas et al., 2010); or, anaerobic ammonium-oxidizing organisms: anammox. Thereby, the execution of batch activity tests can be rather useful to: (i) study the biodegradability of a given wastewater stream (municipal or industrial), (ii) determine the stoichiometric and kinetic parameters involved in the conversion of a specific compound, (iii) study the potential interactions (e.g. symbiosis and competition) between microbial populations and (iv) assess the potential inhibitory or toxic effects of certain wastewaters, compounds or substances.

The nature and type of the batch activity tests can differ depending upon the compounds of interest and the metabolism and physiology of the microbial populations involved in the removal or conversion processes. For instance, they can range from relatively simple aerobic tests where organic matter removal by OHOs is measured to more complex alternating anaerobic-anoxic-aerobic batch tests to assess the activity of PAOs under the presence of different electron acceptors (such as nitrate, nitrite and oxygen) from activated sludge systems performing enhanced biological phosphorus removal (EBPR).

This chapter presents an overview of the most common batch activity tests and protocols and their execution with the aim of assessing the conversion processes involved in: (i) enhanced biological phosphorus removal by PAOs under alternating anaerobic-aerobic conditions, (ii) denitrification via nitrate or nitrite by PAOs, (iii) reduction of sulphate by SRBs, (iv) removal of organics under aerobic conditions by OHOs, (v) denitrification by dOHOs using nitrate or nitrite as final electron acceptor, (vi) oxidation of ammonia and nitrite by AOOs and NOOs under aerobic conditions and (vii) nitrogen removal by anammox bacteria. These experimental protocols aim to serve as a useful guide that establishes a basis for standardizing batch activity tests for use on existing, emerging and innovative treatment processes. It was decided to start the order of presentation with EBPR systems involving PAOs as the processes are complex and include all three biochemical activated sludge environments: anaerobic, anoxic and aerobic.

2.2. ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL

2.2.1 Process description

Enhanced biological phosphorus removal (EBPR) can be implemented in activated sludge wastewater treatment systems by introducing an anaerobic stage at the start of the wastewater treatment lines. High P-removal efficiency, lower operational costs, lower sludge production and the potential recovery of phosphorus have contributed to its application and popularity (Mino et al., 1998; Henze et al., 2008; Oehmen et al., 2007). EBPR is performed by phosphorus (polyphosphate)-accumulating organisms (PAOs) (Comeau et al., 1987; Mino et al., 1998) that, by intracellular accumulation of polyphosphate (poly-P), can remove higher quantities of phosphorus (0.35-0.38 g P g VSS of PAOs) than OHOs (0.03 g P g VSS of OHOs) (Wentzel et al., 2008). The scientific, microbiological and engineering characteristics of the EBPR process have been the main focus of research carried out during the last few decades by different research groups (Wentzel et al., 1986, 1987; Comeau et al., 1986, 1987; Smolders et al., 1994a,b; Mino et al., 1987, 1998; Oehmen et al., 2005a, 2005c, 20i306, 2007; Nielsen et al., 2010). In particular, efforts have focused on developing a better understanding of the actual EBPR metabolic mechanisms, to unravel the microbial identity of the organisms involved, and to optimize the required process configurations, all with the aim of improving and increasing the EBPR process efficiency and reliability.

PAOs are heterotrophic organisms. However, unlike OHOs, PAOs have the unique capability of using intracellularly stored poly-P to produce the required energy (adenosine tri-phosphate, ATP) under anaerobic conditions to store readily biodegradable organic matter (RBCOD), such as volatile fatty acids (VFA) like acetate (Ac) and propionate (Pr), as intracellular poly-phydroxy-alkanoates (PHAs). Stored PHAs are later utilized under anoxic or aerobic conditions for enhanced phosphorus uptake, glycogen synthesis, biomass growth and maintenance. This feature gives PAOs a competitive advantage over other microbial populations of relevance. Thus, PAOs can be enriched to achieve EBPR by recycling activated sludge through alternating the anaerobic and anoxic or aerobic stages, while directing the influent which is usually rich in VFA to the anaerobic stage. A schematic representation of the PAOs' metabolism is shown in Figure 2.2.1.

In the anaerobic stage, PAOs store intracellularly the readily biodegradable organics present in the raw influent or settled sewage (mostly VFA) as PHAs using two other intracellularly stored polymers that take part in the aforementioned metabolism: poly-P and glycogen (a polymer of glucose). Poly-P is hydrolysed and utilized by PAOs to provide the required energy (as ATP) for the transport and storage of VFA as PHAs (Wentzel et al., 1986), while glycogen is used to supply the required reducing power for the conversion of VFA into PHAs as well as to provide the additional required energy (as ATP) (Comeau et al., 1986, 1987; Smolders et al., 1994a; Mino et al., 1998). Thus, the anaerobic uptake of VFA by PAOs results in the storage of PHAs and simultaneous hydrolysis of poly-P and glycogen. The most common PHA polymers stored by PAOs are poly-p- hydroxybutyrate (PHB), poly- -hydroxyvalerate (PHV) and poly-p-hydroxy-2-methylvalerate (PH2MV). Their presence and amount depends on the VFA composition (Ac or Pr). When Ac is the most abundant VFA in the media, PAOs store mostly PHB (up to 90 % of the stored PHAs) (Smolders et al., 1994a), but when Pr is the dominant VFA, then PHAs exist mostly as PHV and PH2MV (Oehmen et al., 2007).

(Continues...)

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