CHAPTER 1
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 plants in general with only a few dominant populations (3–5) being present in a particular plant within the majority of full-scale plants. We should try to avoid the term "species", because a concise species definition is lacking in microbial ecology. Lineages, strains or ecotypes might be equally important for WWTP functioning as "species". The exact microbial community composition in a particular plant depends on wastewater composition, process design, and plant operation, see below. However, in common for most functional groups, the controlling factors determining the community composition is still poorly understood.
Does it matter which bacteria are present in each functional group in a certain treatment plant? This question can in some cases be answered with a clear 'yes', in others 'perhaps' or 'we don't know'. For the filamentous bacteria it is a clear 'yes'. Certain ecotypes cause severe settling properties, others are (in low number) important for sludge flocs as a backbone and thus for the floc structure. A proper identification is essential for the selection of efficient control measures towards the unwanted filamentous bacteria causing settling problems like bulking or foam.
It is more uncertain how important the knowledge about the exact community composition is for the nitrification performance in a certain activated sludge treatment plant, for example. Based on in situ observations it has been suggested that the presence of several lineages of ammonia and nitrite oxidizers ensures a more robust and stable system (e.g. Daims et al., 2001c) compared to the presence of only a single lineage from each functional group. But it is less documented what the exact community composition of a certain functional group means for plant stability and operation. Future studies, all based on a reliable identification of the microbial populations, for example by FISH, will show. A related question is whether strain-level microbial diversity influences process stability in WWT. For instance, very closely related nitrite oxidizers, which are slightly different on the genome level, may co-exist in the same plant. Due to a high sequence similarity (or even identity) of their 16S rRNA, the diversity of these strains would easily be overlooked by current approaches using methods such as FISH. However, the genomic differences may result in a biologically significant variety of phenotypes, which respond differently to events such as changes in wastewater composition or bacteriophage attack. With the latest molecular methods such as environmental genomics and deep DNA sequencing at hand, future research will elucidate the importance of such microbial microdiversity from an applied perspective.
Only a few comprehensive studies have been published describing the total community composition from full-scale wastewater treatment plants. They deal with activated sludge plants, such as industrial plants for C-removal or N-removal (Juretschko et al., 2002) or a plant with biological N- and P-removal from a mixture of domestic and industrial wastewater (Kong et al., 2007). However, several studies of specific populations have been carried out in various full-scale plants, referring solely to nitrifiers, denitrifiers or filamentous bacteria, for example. These studies are briefly described in the specific chapters. A summary of the most commonly observed species and genera encountered in WWTPs is shown in Table 1.1.
1.3 FACTORS OF IMPORTANCE FOR THE GROWTH OF MICROORGANISMS
The species composition in treatment plants depends on wastewater composition, process design, and plant operation. It is obvious that certain functional groups are dominant only if specific processes are included in the plant's process design (e.g. N-removal or EBPR). As mentioned above, the controlling factors determining the species composition are still poorly understood for many species, so during studies of the microbiology in WWTPs it is important to observe and register the potential factors that may be decisive for the presence of the different species. An overview of such factors is given in Table 1.2 and more advice on these is given elsewhere (e.g. Wilderer et al., 2002).
Most important for activated sludge plants is whether solely C-removal is included in the plant design – meaning that only aerobic tanks are present (besides clarifier), or whether denitrification/EBPR are also included, meaning that anoxic/anaerobic tanks are present in addition to the aerobic ones. The selective pressure due to anoxic/anaerobic tanks substantially changes the population structure. Sludge age (mean cell residence time), which is determined by the sludge loading, is also extremely important. A low sludge age (<5–10 days) may not allow nitrification to occur due to the low growth rates of the nitrifiers, whereas a high sludge age (>20–30 days) is important to obtain full N- and P-removal in temperate climates. Treatment plants running at very high temperatures (>40–C), such as those treating special industrial wastewater, often select for unusual microbial communities.
The composition of the incoming wastewater is another decisive factor for bacterial growth. Industrial wastewater is often less complex than municipal wastewater, meaning fewer microorganisms may dominate in the treatment plants. Furthermore, the soluble fraction is often higher. Industrial wastewater may also not include important nutrients, such as P or N or other micronutrients. Domestic wastewater is usually more complex with a high fraction of particulates and a more balanced ratio of organics and nutrients. Other important factors are temperature, salinity, presence of toxic substances, and pH value. Likewise, the incoming microorganisms may affect the population structure in the treatment plant.
The operation of the plant may also affect the population structure. It is closely interrelated to the process design of the plant. The exact operation of anoxic/anaerobic mean cell residence time, oxygen concentration in aerobic tanks, addition of chemicals, carbon sources, and many other factors may affect the population composition.
The importance of the technology platform being applied to carry out a specific process, such as nitrification, is also poorly understood. Do we get the same nitrifiers in a full-scale plant based on activated sludge with different process configurations (e.g. sequencing batch reactors, continuously stirred reactors or plug-flow reactors), biofilm reactors (e.g. upflow or downflow biofilters, airlift reactors, granules reactors), or membrane bioreactors treating the same wastewater? Few studies have investigated this in detail, but the general impression is that we are often dealing with the same species/groups, although perhaps with slightly different strains/ecotypes (see also above). More studies based on a reliable identification of the microbial populations are needed.
1.4 THE USE OF THIS FISH HANDBOOK
This handbook contains a detailed description of the FISH protocol for identification and quantification of various bacteria typically encountered in biological wastewater treatment. The bacteria included cover several functional groups: nitrifiers, denitrifiers, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), filamentous bacteria involved in bulking or foaming, and some others. They can be found in aerobic or aerobic/anoxic/anaerobic treatment systems based on activated sludge or biofilms applied in various technologies. We have not included bacteria present in digesters (anaerobic digestion), fuel cells or bacteria carrying out more rarely encountered treatment processes such as treatment of S-containing waste (for So production) or removal of specific pollutants (e.g. from polluted sites). Some of these groups may be included in future editions of this book.
The handbook does not cover protozoa as the molecular methods are still not ready for a proper detection of these.
Detailed information about the ecophysiology, ecology or management of the different bacteria is outside the scope of this handbook. Such information can be found in the specific literature, manuals, and books (see the different chapters) or in the new IWA book on "Microbial Ecology of Activated Sludge" (Eds. Seviour and Nielsen, 2009).
For each functional group an overview of the identities of the bacteria and their abundances is presented. Extensive tables describe the gene probes to be applied for the detection of the microbes, and phylogenetic trees show the coverage of the various probes. Furthermore, we have included a description of the typical morphologies targeted by the probes and many of these are documented by FISH images in the color figure section (Chapter 9). Based on our own experience we recommend the most suitable probes. Most probes are selected on the following criteria: they were designed based on published fulllength sequences, the probes are published, and details can be viewed in probeBase (www.microbialecology. net/probebase). Other probes are briefly mentioned in the text. They might be relevant in special cases.
New or improved gene probes for detection of relevant microbes are continually being developed, so we plan ongoing revisions of this book, the first in 1–2 years. Information about new editions and other relevant updates can be found on the web page of the IWA specialist groups (www.iwahq.org). This handbook has been developed in conjunction with the IWA specialist group on 'Activated Sludge Population Dynamics'.
Errors, suggestions for inclusion of other probes, important experiences or other comments are more than welcome as they can be beneficially included in future editions. Please e-mail Per Halkjær Nielsen (phn@bio.aau.dk).
