Introduction

Per Halkjær Nielsen, Holger Daims, and Hilde Lemmer

1.1 IDENTIFICATION OF MICROORGANISMS IN ACTIVATED SLUDGE AND BIOFILMS

Until very recently, culture-dependent methods such as plate count or Most-Probable Number (MPN) counting have widely been used for enumeration and detection of bacteria being relevant to biological wastewater treatment performance. In fact, such standard methods are in many cases still used for effluent quality control, particularly with respect to pathogens and various indicator organisms (e.g. APHA Standard Methods). However, today we know that these methods suffer from severe limitations as from all types of microbes in environmental samples (also pathogens), only a very small fraction is cultivable on media generally applied. Therefore, this approach is prone to lead to serious misinformation. Thus, we strongly advocate for a change to using culture-independent molecular methods in all sorts of microbiological investigations in wastewater treatment plants (WWTPs).

Among the cultivation-independent methods for detection, fluorescence in situ hybridization (FISH) with ribosomal RNA (rRNA)-targeted probes (gene probes) is a very powerful tool for identification of microorganisms in activated sludge and biofilm biocenoses from WWTPs. This method is described in detail in Chapters 7 and 8 of this book. Most known functional key microorganisms in wastewater systems can be reliably identified and quantified by this method.

Furthermore, other molecular methods exist, primarily PCR-based methods. Quantitative PCR (q-PCR) is now getting more commonly applied in environmental samples, but the method has several drawbacks compared to FISH. This is due to biases concerning nucleic acid extraction, the PCR reaction, and also the fact that PCR-based approaches do not quantify microbial cells, but measure copy numbers of marker genes. DNA microarrays carrying rRNA-targeted probes, so-called "phylochips", have a great potential as high-throughput tools for the qualitative detection of hundreds or even thousands of different uncultured microbes in only one experiment. When combined with autoradiography, phylochips become "isotope arrays" useful to track functional traits of microbes such as nitrifying bacteria in WWTP (Adamczyk et al., 2003). However, to date no quantitative phylochip-based assay exists that would allow for observing shifts in the abundances of probe-target microbial populations. This limitation is due to technical problems, such as saturation effects during hybridization, with the microarrays which are very difficult to overcome.

In contrast to the other methods, by using FISH it is possible to observe the morphology and to quantify numbers of bacteria or the equivalent biovolume. Thus, in our opinion, FISH is for the time being the method of choice for detection and quantification of microorganisms in WWTP as detailed in Chapters 7 and 8.

A special case is the identification of filamentous bacteria. These have primarily been identified based on their morphology and simple staining techniques using light microscopy since their first comprehensive description by Eikelboom (Eikelboom, 1975). Several manuals have since been published (e.g. Eikelboom 2000; Jenkins et al., 2004), all of them being based on his original work. However, today it is clear that although some filamentous bacteria can be fairly reliably identified in this way, the majority can not. As described in Chapter 5, we strongly recommend to also apply FISH for the identification of filamentous microorganisms, after having accomplished a preliminary morphological identification using the manuals.

1.2 THE MICROBIOLOGY OF BIOLOGICAL WASTEWATER TREATMENT

Biological treatment of municipal and industrial wastewater worldwide is primarily carried out by the activated sludge (AS) process. New technologies are being developed such as biofilm reactors, membrane bioreactors, sequencing batch reactors, etc., but they basically all derive from the AS process. The common purpose of all these technologies is the use of microorganisms to remove carbon (C), nitrogen (N), phosphorus (P), micropollutants and pathogens.

New interesting more sustainable solutions are appearing. They include for example recovery of nutrients (e.g. P) from wastewater, or conversion of organic waste components to usable, valuable compounds such as bioplastics (polyhydroxyalkanoates, PHA). Conversion of organic waste to energy by methane production during anaerobic digestion has been utilized for decades and these processes are further being developed together with other energy yielding processes such as microbial fuel cells.

Management of these complex microbial systems (or 'microbial resource management' for the new sustainable solutions, Verstraete et al., 2007) relies on a fundamental knowledge about the microbial populations being involved and about the factors that regulate their activity. A reliable identification of the microorganisms involved is fundamental and with the today's toolbox of various culture-independent methods is possible with a high sensitivity and precision. Not only the identity, but also knowledge about their ecophysiology, ecology, and population dynamics is essential. The present methodological approaches range from single cell microbiology (e.g. microautoradiography and FISH-Raman microspectroscopy; Huang et al., 2007), expression of specific functional genes, systems biology (genomics, transcriptomics, and proteomics) to lab-scale reactors and full-scale studies of chemical transformations. In this way we are gaining a rapidly increasing understanding of key microorganisms being involved in many processes and how to affect their presence and activity. However, there is still much to learn about full-scale systems since most studies so far have been carried out in lab-scale and pilot-scale reactors.

Several functional groups of bacteria being involved in the most common treatment processes are now fairly well identified and described. It is primarily bacteria involved in nitrification and to some extent those involved in denitrification, many bacteria involved in the enhanced biological P-removal (EBPR), and most bacteria causing settling problems (bulking) or foam/scum formation. In each functional group, for example the nitrifiers, a limited number of phylogenetic lineages (<10) is encountered in nitrifying...