CHAPTER 3
Protozoa in Wastewater Treatment: Function and Importance Wilfried Pauli 1, Kurt Jax 2, Sandra Berger 1 1 2
Institut für Biochemie und Ökotoxikologie, Freie Universität Berlin, Ehrenbergstr. 26–28, D-14195 Berlin, Germany, E-mail:
[email protected] Zentrum für Ethik in den Wissenschaften, Universität Tübingen, Keplerstrasse 17, D-72074 Tübingen, Germany
Protozoa constitute a major link between the highly productive and nutrient retaining microbial loop and the metazoans of the classical food web. Protozoa are efficient at gathering microbes as food, and they are sufficiently small to have generation times that are similar to those of the food particles on which they feed. They are, in quantitative terms, the most important grazers of microbes in aquatic environments, balancing bacterio-plankton production. Protozoa not only play an important ecological role in the self-purification and matter cycling of natural ecosystems, but also in the artificial system of sewage treatment plants. In conventional plants ciliates usually dominate over other protozoa, not only in number of species but also in total count and biomass. It is generally accepted that their feeding on bacteria improve the treatment, resulting in a lower organic load in the output water of the treated wastes. Due to their biodegradation potential some attempts have been made to use ciliates specifically in environmental biotechnology. As biosensors they could provide valuable information regarding adverse effects of environmental chemicals on this part of the biocoenosis essential for the effective operation of biological waste-water treatment processes. Keywords. Protozoa, Ciliates, Ecology, Sewage treatment, Environmental biotechnology
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Ecological Role of Aquatic Protozoa with Special Regard to Ciliates Within the Microbial Food Web . . . . . . . . . . . . . 205
1.1 1.2 1.3 1.4
Introduction . . . . . . . . . . . . . . . . . . . . Traditional Food Webs and Microbial Food Webs The Role of Protozoa in Aquatic Food Webs . . . Outlook . . . . . . . . . . . . . . . . . . . . . . .
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2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.5.1
Background . . . . . . . . . . . . . . . . . . . . . . . Wastewater . . . . . . . . . . . . . . . . . . . . . . . Biological Treatment Processes . . . . . . . . . . . . Bacterial Biofilms . . . . . . . . . . . . . . . . . . . . Activated Sludge . . . . . . . . . . . . . . . . . . . . Protozoa in Biological Wastewater Treatment Plants Occurrence . . . . . . . . . . . . . . . . . . . . . . . Species Composition . . . . . . . . . . . . . . . . . . Plant Specific Basic Communities . . . . . . . . . . . Biomass . . . . . . . . . . . . . . . . . . . . . . . . . Ecological Framework . . . . . . . . . . . . . . . . . Sludge Loading . . . . . . . . . . . . . . . . . . . . .
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The Handbook of Environmental Chemistry Vol. 2 Part K Biodegradation and Persistence (ed. by B. Beek) © Springer-Verlag Berlin Heidelberg 2001
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2.2.5.2 2.2.5.3 2.2.5.4 2.3 2.3.1 2.3.2 2.3.2.1 2.3.2.2 2.3.2.3 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH-Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O2-Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of Protozoa for Wastewater Treatment . . . . . . . . Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reduction and Elimination of Suspended Particles and Bacteria Clearing Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Findings . . . . . . . . . . . . . . . . . . . . . . . . “Field”-Observations . . . . . . . . . . . . . . . . . . . . . . . . . Elimination of Dissolved Substances . . . . . . . . . . . . . . . . Flocculation and Composition of the Bacterial Community . . . Reduction of the Total Biomass . . . . . . . . . . . . . . . . . . . Influence of Protozoa on Bacterial Metabolism . . . . . . . . . . Filamentous Bacteria and Protozoa . . . . . . . . . . . . . . . . .
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Biodegradation Potentials of Ciliates . . . . . . . . . . . . . . . . . 243 Ciliates as Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . 245
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List of Abbreviations BOD(5) COD dw fm
biological oxygen demand (index: within 5 days) chemical oxygen demand dry weight sludge loading [g BOD (g MLSS day)–1 or g BOD (g MLVSS day)–1], also known as “food to micro-organism (F/M) ratio” F/M-ratio see fm MLSS Mixed-liquor suspended solids, sludge solids (g m–3; concentration of the suspended solids in an aeration tank including inorganic matter) MLVSS Mixed-liquor volatile suspended solids (g m–3; corresponds to the organic, i.e., combustible content of the sludge, which amounts to ca. 70% of the sludge solids: 0.7 MLSS≈MLVSS; this parameter is often used as indicator of microbial concentration, although it does not distinguish between biochemically active material and inert or dead material in the sludge) EC/LC50 50% effective and lethal concentration, respectively
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1 Ecological Role of Aquatic Protozoa with Special Regard to Ciliates Within the Microbial Food Web 1.1 Introduction
There is hardly any place on earth in which protozoa cannot be found. They are abundant in terrestrial as well as in aquatic systems. In the latter they are present in high numbers of species and individuals both in the oceans and in freshwater habitats. Some taxa live attached to solid substrates or within the sediment, some as part of the plankton. An overview of the data about the abundance of protozoa in aquatic habitats gives a first indication that these organisms are not negligible in aquatic environments – although in fact they are still often neglected. In the plankton of highly productive lakes, densities of small flagellates (< 20 mm body size) of more than 106 cells per ml were reported [1] and in studies on the periphyton of small bodies of waters maximum values of more than 1350 cells per cm2 of the much larger testate amoebae specimens were encountered [2]. However, these numbers do not make any statements about the ecological interactions in which the species are involved and the role they play within those processes which mostly are seen as the essence of ecosystem dynamics, namely the fluxes of energy and material. It is the objective of this paper to provide a short introduction to the current knowledge of these roles as regards aquatic environments. 1.2 Traditional Food Webs and Microbial Food Webs
Traditionally, food webs in aquatic systems were illustrated as in Fig. 1. Going back to the limnologist August Thienemann, the different species within a body of water were characterized by the categories of producers, consumers of different order (primary consumers, secondary consumers and so on) and decomposers [3]. The latter live on the dead organic matter and mineralize the organic compounds to inorganic nutrients, e.g., phosphorus, nitrogen, etc. These categories were also the basis on which Raymond Lindeman [4] built his famous trophic dynamic concept of ecology which was the first implementation of Arthur Tansley‘s ecosystem concept [5]. Energy enters the system as light and is processed as organic matter along the food chain or food web until most of the energy is dissipated by respiration. In aquatic habitats these functional categories – trophic levels in Lindeman’s parlance – were commonly attributed to phytoplankton (producers), zooplankton (primary consumers), and different kinds of vertebrates on the higher trophic levels. Protozoa and particularly bacteria were seen as decomposers, mainly restricted to sediments and other surfaces, but of minor importance in the pelagic food web. This association of bacteria and protozoa with decaying matter was recognized and used for applied purposes rather early. Protozoa were used as bioin-
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Fig. 1. Diagram of the “classical” food web in lakes. Modified, according to [6]
dicators for the saprobic states of natural and manmade freshwaters as early as 1908 (e.g., [7, 8]). Their dynamics in the process of decomposition of organic substances were clarified by the middle of the century. Meanwhile, classical studies on this topic were made by Bick and co-workers (e.g., [9, 10]) who investigated the succession of micro-organisms, in particular ciliated protozoa, in the course of the “self-purification” of water enriched with sewage and other organic substances. However, during the last two decades there have been some new insights which have broadened and fundamentally changed our way of looking at the water of lakes and oceans and which affect the role protozoa and other microorganisms are supposed to play within aquatic systems. These insights were initiated by the appearance of some new actors on the stage of the ecological theater which also radically changed the roles in which protozoa were perceived. In 1974 Pomeroy [11] presented a paper in which he developed new ideas about the interactions of the pelagic organisms. Although these ideas were first developed in connection with marine systems they were soon transferred to freshwater habitats. The main point made is that, besides and connected with the classical “macroscopic” food web, there exists a microbial food web. The reason why these microbial food webs were discovered so late can, to a high degree, be attributed to the development of new methods in aquatic ecology.
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By the early 1970s it was recognized that an important part of the pelagic organisms had been neglected as a result both of the methods used and of the theories regarding interactions in the water. Using direct counts of bacteria with epifluorescence methods instead of plate counts, it turned out that the abundance of bacteria in the open water had been underestimated by orders of magnitude. Only 0.1–1% of the actual abundance had been counted [12]. Furthermore, most investigations of marine and freshwater plankton used plankton nets with a mesh size of 20 mm or even 60 mm, while all smaller organisms were thought to be of minor importance. Finally, the methods of conserving planktonic protozoa were inadequate and even larger protozoa were neglected or underestimated as components of the pelagic species assemblages [13]. What was collected and counted were those fractions of the plankton which we now call the micro- and macroplankton, i.e., organisms bigger than 20 mm (Table 1). Thus, not only all smaller organisms, the pico- and nanoplankton – consisting of bacteria, Cyanobacteria, small protozoa, and small eukaryotic algae [14] – but also many larger protozoans were to a large extent excluded from the quantitative sampling. However, it turned out that especially this small sized fraction of the plankton is of extreme importance in terms of energy- and material fluxes. New measurements revealed that the major part of the metabolic activity in plankton was displayed by the size fraction below 10 mm [15]. The most productive component of the pelagic food webs was not, as thought earlier, the planktonic eukaryotic algae of the microplankton, but the tiny Cyanobacteria, mostly of the genus Synechococcus, and some small eukaryotic algae. The percentage of primary production in terms of carbon varies between 1% and 90% in marine waters – with higher ratios in more oligotrophic conditions – and 16–70% in fresh waters [16]. For oligotrophic lakes 50–70% are documented, while the autotrophic picoplankton amounts to 10–45% of the total phytoplankton biomass (standing stock, measured as chlorophyll) [17]. Data for marine habitats give estimates of 20–80% [18]. Similarly, the abundance of heterotrophic picoplankton, i.e., heterotrophic bacteria, is much higher than previously thought and can approach 109 cells in highly eutrophic fresh waters [1]. However, the new theory incorporates some new links rather than just adding picoplankton to the classical food web. Figure 2 presents a very simple diagram of what a microbial food web might look like, given the current status of knowledge. Table 1. The size classes of planktonic organisms
Picoplankton
Nanoplankton
Microplankton
Macroplankton
0.2–2 mm Bacteria Cyanobacteria Algae Rhizopods Flagellates Ciliates
2–20 mm Algae Flagellates Ciliates Ciliates
20–200 mm Algae Rhizopods Crustaceans Rotatoria Nauplii
> 200 mm Ciliates Rotatoria Fish larvae
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Fig. 2. The food web of the lake plankton. The classical food chain (open circles) is supplemented by the elements of the microbial loop (filled ovals and square). DOM: dissolved organic matter
The earlier food chain from algae via macrozooplankton to fish still exists but is supplemented by a new section which is commonly called the microbial “loop.” This consists of the picoplankton (“algae,” i.e., Cyanobacteria and heterotrophic bacteria), protozoa, and a compartment of non-living material, i.e., dissolved organic matter (DOM). DOM is lost and excreted in substantial amounts by both algae and Cyanobacteria and constitutes the energy source for the heterotrophic bacteria. The rate of fixed carbon lost by phytoplankton cells may vary between 10% and 40% depending on the physiological status of the cells [13]. The picoplankton is grazed by protozoa which themselves are preyed upon by the metazoan zooplankton, thus coupling the microbial loop to the traditional parts of the food web. As cells with a size of up to 2 mm hardly get lost through sedimentation, the microbial loop not only adds some new links to the classical food web but keeps the nutrients (DOM and inorganic nutrients) within the water body and minimizes losses to the deeper, non-productive regions of the waters or even the sediment. This seems to be particularly important during the summer stratification of oligotrophic lakes, in which the epilimnion, the upper and photosynthetically active region of the lake – the euphotic zone – is temporarily cut off from the richer nutrient supply of the deeper waters [17]. 1.3 The Role of Protozoa in Aquatic Food Webs
From this scheme the new role of protozoa within the food webs of aquatic systems seems obvious. They are not only – in the same way as bacteria – decomposers associated with the decay of organic material, but they are a link between
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the highly productive and nutrient retaining microbial loop and the metazoans of the classical food web. Most microplankton organisms are unable to utilize particles smaller than 5 mm directly [18]. Protozoa “repack” the organic material into edible portions and thus make it available to crustaceans, rotatoria, and other metazoans. There is empirical evidence that planktonic protozoa graze effectively on picoplankton and also that protozoa constitute a valuable diet for crustaceans [19]. Thus both necessary links between picoplankton and metazoa have been established. The details of the microbial webs, however, are still the subject of research and discussion. The specific pathways and the number of steps over which energy and nutrients are transferred are subject to much variation. There is temporal variation, e.g., seasonally, [20] and there is spatial variation both within lakes and even more if different lakes are compared. The compartment of protozoa can be divided in several ecological relevant ways. Not only is there a taxonomic division between flagellates and ciliates, but also a physiological one, relating to the nutritional mode (autotroph, heterotroph, mixotroph, etc.) which does not correspond with the classic taxonomic or “trophic level” boundaries [21]. Furthermore the body sizes of the different taxa are important features for their position within the food webs. In many cases bacteria are grazed upon mainly by small heterotrophic flagellates, the heterotrophic nanoplankton (HNAN), which in most cases turned out to be the most efficient predators of bacteria that were able to control the bacterial populations even during their highest productivity (e.g., [1, 22]). Berninger et al. [1] found a clear correlation between the abundance of bacteria and HNAN in comparing samples from more than hundred freshwater sites of different trophic states. The numbers of the two groups of organisms differed by two or three orders of magnitude, with maxima of more than 106 specimen of HNAN and 109 specimens of bacteria per ml. They inferred predator-prey relationships between these groups. HNAN are sometimes grazed upon directly by metazoa, while in other bodies of water ciliates constitute the main predators [17, 23]. Heterotrophic flagellates, possessing high turnover rates, inhabit a central position in the transfer of organic carbon in most microbial food webs. But what about the ecological roles of ciliates? In some cases, especially in productive waters, ciliates can also graze effectively on picoplankton and can even be the most important bacterivores, taking a key position for the transfer of matter to the metazoan links [23]. However, smaller bacterivorous ciliates with high grazing efficiencies need a threshold abundance of bacteria to persist on this diet. Beaver and Crisman [24] gave an estimate that small ciliates (20–30 mm) were “largely excluded from lakes having 50 mm), being mainly phytophagous and grazing on nanoplanktic algae, dominate the ciliate assemblages in oligotrophic lakes, with low bacterial abundance. Mixotrophic ciliates with endosymbiotic algae can even contribute substantially to pelagic autotrophic biomass in some lakes (15% of annual total [25]). The overall number of planktonic ciliates in lakes is correlated with the trophic state of the water bodies.While under oligotrophic conditions abundancies
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of 3–10 cells ml–1 were recorded, 90–215 cells ml–1 were recorded in hypereutrophic waters [25]. The length of the food chain originating from bacteria and Cyanobacteria and the identity of links involved is important to the still unresolved question as to whether the microbial loop is acting as a link or a sink for organic material. Adherents of the latter position argue that a microbial food chain with four steps will be unlikely to transfer any substantial amount of organic carbon to the metazoan part of the web [15, 26, 27]. The answer to this question is dependent on several variables. Besides the trophic states of the waterbodies, other abiotic variables such as temperature and acidity are relevant for the specific patterns of the microbial web [25] and also the species composition of the whole food web [28]. In some cases organic material is transferred from picoplankton via heterotrophic flagellates to larger ciliates and then to crustaceans or other metazoans. In other cases crustaceans may directly feed on nanoplankton, while ciliates are of minor importance [29]. Even though most metazoans cannot feed effectively on small particles of the order of few mm, some freshwater species, in particular cladocera of the genus Daphnia, can effectively control bacterial abundance (although they may not persist on bacteria alone), thus shortcutting the microbial loop [17, 28]. The presence or absence of a single species can thus change the pathways completely, deciding the coupling or decoupling of the microbial loop from the metazoan web. The proportion to which different groups of organisms contribute to different nutritional types in a lake is also seasonally variable [17, 20, 28]. In this regard, the scheme displayed in Fig. 3 comes closer to the perceived processes than many other representations, in that a multitude of pathways is possible which may be more or less important at different times.
Fig. 3. Diagram of the food web in lake plankton. In contrast to the scheme in Fig. 2, the compartment of protozoa has been differentiated. Note that not all pathways are realized at any one time. See also text. DOM: dissolved organic matter
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As mentioned above, the microbial loop is not only important for the transfer of energy in the form of organic carbon, but also for the cycling and retention of nutrients. This is especially important in oligotrophic situations, where nutrients like phosphorus and nitrogen are scarce – at least during certain times of the year. The phosphorus dynamics of the pelagic zone seem to be strongly determined by the interactions of algae, bacteria, and protozoan grazers. Algae and bacteria compete for P, with bacteria being more efficient in the uptake of P. Bacterial grazing by protozoa was demonstrated to enhance phosphorus turnover and mineralization [30]. As grazed bacteria populations grow faster their excretion of P also becomes stronger. Furthermore, protozoan grazers increase the amount of organic P by excretion, which seems to be of special importance for phytoplankton [31]. Although this compound is also excreted by micro- and macrozooplankton, the high metabolic rate of protozoa leads to higher excretion rate of this group of organisms. Buechler and Dillon [32] estimated that if ciliates only contribute 1% to the biomass of a zooplankton assemblage, they should be able to contribute 50% to the release of dissolved P. A similar situation exists with regard to nitrogen in cases where nitrogen is a limiting factor for the growth of algae and bacteria. Bacteria can also outcompete phytoplankton for N and thus serve as a sink for nitrogen within the food web. However, as has been demonstrated experimentally, the presence of bacterivorous protozoan grazers leads to a partial remineralization of N and allows an increase in algal biomass [33]. The degree to which this process is of importance depends on the carbon available for the bacteria. As Caron et al. [33] concluded: “the role of bacterivorous protozoa as mineralizers of a growthlimiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high”. 1.4 Outlook
Most of the interactions described above were investigated in the pelagic part of aquatic habitats. However, as mentioned above, many protozoa are closely related to surfaces within the water bodies, be they sediments, plants, and stones, or even microscopic aggregates within the pelagic zone. In lakes or oceans the main metabolic activity is certainly associated with the pelagic zone. Regarding streams or small water bodies, the surface-related biota gain in importance for the fluxes of energy and materials. In streams, a true plankton only exists in the slow flowing lower reaches of large rivers. Thus, most organismic activities are found in and on the benthic parts. Many of the aspects discussed above will also be valid in these environments. However, there will surely be differences. Although some data is available on the numbers and production of protozoa in these microhabitats [34–36], our understanding of the complex web of interrelations is much less than for the open water. To a considerable degree this seems to be a consequence of the methodical difficulties. Benthic assemblages are highly heterogeneous in space and time and this heterogeneity, i.e., the small scale spatial arrangement of the different components, is by itself of importance for the nature of the interactions between protozoa and the other parts of these
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assemblages. Thus we are only just beginning to delve deeper into the complicated patterns and dynamics of those biofilms. There is now important evidence that these biofilms are also highly productive but also very retentive in regard to nutrients [37]. Nutrient pulses are retained much longer within the periphyton assemblages of streams than would be expected on the basis of a continuous water flow. There are certainly many other important ways in which protozoa are involved in the ecology of aquatic systems. For example, little is known about informational relations between protozoa and other members of the species assemblages, although there may be indications in this direction (e.g., [38]). Also, our view of microbial food webs may change during the next years with the new awareness that even the pelagic zone of lakes is not as homogenous as it seems at first sight. In addition to rather macroscopic stratifications of abiotic factors and the related stratifications of organisms, the role of tiny and – in the realm of human time-scales – fleeting aggregates of small detritus particles, bacteria, protozoa and algae come into prominence, the so called “lake snow.” These aggregates may turn out to be hot spots of microbial activity, and especially for the grazing activities of protozoa. There are data that indicate that ciliate bacterivory is especially high in lakes with high amounts of suspended organic matter [39]. Similar to biofilms on solid substrates, the microenvironment on, in, and around these aggregates can be chemically strangely different from the average water column data. It remains to be seen, what these new insights will bring about for the understanding of the ecological processes in freshwater habitats.
2 Protozoa in Wastewater Treatment 2.1 Background 2.1.1 Wastewater
Wastewater includes municipal, industrial, and agricultural wastewater as well as rainwater. The relative proportions of wastewater for West Germany (1980) were 32% municipal, 47% industrial, and 1% agricultural wastewater, plus 20% rainwater run-off in areas with main drainage. All wastewater produced in towns and communities is termed municipal sewage. This expression covers domestic wastewater (50%), extraneous water (leachates 14%), and wastewater from industry and commerce (36%) [40]. Municipal sewage is treated as follows: – Initial mechanical purification or sedimentation – Biological purification or clarification – Further purification, e.g., elimination or reduction of the nitrogen, sulfur, or phosphate content, polishing, filtration – The treated wastewater is then discharged into the receiving stream (Fig. 4)
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Fig. 4 a– c. Types of common sewage treatment plants – flow diagram of: a activated sludge plants; b, c biofilm processes (trickling filter and Rotating Biological Contactor, RBC, respectively). In the activated sludge process (a) the wastewater is exposed to a mixed microbial population in the form of a flocculent suspension. In fixed medium systems the wastewater is brought into contact with a film of microbial slime (b) on the surfaces of the packing medium, (the wastewater trickles through the bed, most commonly consisting of stacked stones), or (c) on a partly submerged support medium which rotates slowly on a horizontal axis in a tank through which the wastewater flows
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Table 2. Average contribution of settleable (sedimentation within 2 h) and non-settleable
matter and their respective biochemical oxygen demand (BOD5) to the total organic load of municipal sewage, according to [157] Organic load (in total ca. 450 mg/l)
Æ Æ
Settleable: 33% (w/v) or 150 mg/l, 33% (BOD) Non-settleable: 67% (w/v) or 300 mg/l, 67% (BOD)
– Æ Æ
Dissolved: 83% (w/v) or 250 mg/l, 75% (BOD) Suspended: 17% (w/v) or 50 mg/l, 25% (BOD)
All substances present in sewage are classified according to their significance for wastewater treatment plants. Organic content is of particular importance for degradation processes. It is quoted in terms of the chemical or biochemical oxygen demand (COD, BOD) of the organic substances. Furthermore, a differentiation is made between suspended and dissolved wastewater components. Approximately two-thirds of the total load (organic and inorganic) of municipal sewage is in solution. With regard to the organic load almost half is in solution, the rest consists of colloidal material (25%) or is bound to particles which sediment (75%). Similarly, about half of the oxygen demand of biochemically degradable organic compounds is attributed to the dissolved fraction, of the other half one third to floating and two thirds to particulate matter. After a 2 h sedimentation period, two-thirds of the total organic load remains in the supernatant (also two-thirds of the total BOD). About 25% of the dissolved organic load is bound to colloids and particles which do not sediment (Table 2). Carbohydrates are not usually present in municipal wastewater plants. They are metabolized on route in the sewage. Proteins are also hydrolyzed in the sewers. The main task of the wastewater treatment plant is then to eliminate fatty acids and the amino acids formed by protein hydrolysis. Municipal sewage averages an organic load of 300 mg BOD5 l–1 (ca. 450 mg l–1 organic content). Activated sludge plants aim for effluent values < 20 mg BOD5 l–1, i.e., a reduction in the organic content of more than 90% [41]. For industrial – as opposed to municipal – wastewater, no generalizations can be made regarding type and amount of load. Diverse organic and inorganic loads are produced by different industrial sectors. Even within a sector values vary according to the production methods and environmental requirements. Wastewater from the chemical industry often exhibits toxic or inhibitory effects. 2.1.2 Biological Treatment Processes
It is well known that a microbial degradation of organic substances takes place in natural flowing waters. This natural, self-purifying capacity of water became overtaxed by the increase in population and industrialization. Attempts were then made to pre-treat partially or fully sewage by mechano-biological processes, before discharging it into the surface water.
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A conscious use of biological degradation began after bacteria were discovered in the nineteenth century. Two principles were implemented: activated and fixed-bed processes. The latter have been in use since 1882 and utilize the slime growth of organisms in the receiving stream. The activated sludge process, which takes advantage of the self-purification properties of the suspended organisms in the receiving water body, was developed in 1913, and the first German plant was operational in 1926 [42]. Both methods are still in use today. In Germany the activated sludge technique has taken precedence, due to its higher performance capacity, particularly for extended wastewater treatment including nutrient elimination. However fixed-bed reactors in combination with activated sludge techniques are finding increased application today. As submerged aerators they increase the active biomass and the age of the sludge in activated sludge plants, making a positive contribution to the purification efficiency [43]. The underlying principle of biological wastewater treatment is to transform the majority of dissolved and suspended substances into biomass which can then be removed either by sedimentation (activated sludge) or by fixing (submerged aerator contactors). In this way, a nutrient concentration exceeding the degradation capacity of local surface waters, resulting in disruption or even destruction of natural biological systems, can be avoided: Direct discharge of substances would result in anaerobic or aerobic burdening of the sediment of surface waters; high oxygen consuming, organic content (BOD5) in the effluent can overtax the oxygen household of the water, through its rapid conversion by heterotrophic organisms; direct discharge of plant nutrients, particularly nitrogen compounds and phosphates, encourages algal growth, with negative effects on the water (larger pH- and O2-fluctuations, sludge formation). At the same time, however, the discharge of bacteria – used for the fixation of wastewater substances – should be kept to a minimum. All biological processes have in common that they involve sectors of natural metabolic cycles. In wastewater treatment plants, the only difference from natural processes is that part of the reaction chain is technically controlled. The performance is dependent not on one specific species with a high degradation capacity, but on the interaction of a wide range of different organisms. Over the last 20 years the traditional model of a vertical material and energy flow, starting from nutrients through to decomposers and primary producers and both primary and secondary consumers, has been replaced by a more complex ecological web, which takes into account the network of microbial systems and their significance for turnover of matter (see Sect. 1.2). In treatment plants, due to the high organic content of the wastewater, a biocoenosis of organisms forms, primarily made up of members of the group of decomposers, i.e., saprophytic bacteria. The majority of the bacteria degrade dead organic matter, in the presence of oxygen, to carbon dioxide and water. Nitrogen is released in the form of ammonia. Bacteria are significant in wastewater treatment due to their large surface area in relation to their body volume and their associated high metabolic and reproductive rates. Apart from these prokaryotic forms of life, protozoa (unicellular, animal organisms) are the next most important group of organisms in the wastewater biocoenosis. Together
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with bacteria they form a closely related microbial system which forms the basis of the so-called natural self-purification process. 2.1.3 Bacterial Biofilms
In both fixed-bed and activated sludge processes, microbial biofilms – either as slime growth or flocs – are fundamental for the turnover of organic waste. The colonization of surfaces by bacteria is a widespread process in the environment. In natural biotopes, bacteria favor the colonization of suspended particles and sediment. By far the majority (99%) of all bacteria in the environment adhere to surfaces such as stones, sediment, and soil. Important physico-chemical processes, forming the basis for the biomass layer, precede the attachment of a biofilm. Dissolved organic molecules (polysaccharides, proteins, humic acids) accumulate spontaneously on the surface of very different materials forming a “conditioning film,” on which bacteria colonization follows. The cells are immobilized and produce extra-cellular polymeric substances which anchor the organisms to the surface and to each other. Embedded in this matrix, microbial communities of complex composition are built up, usually in several layers. Biofilms are not static systems, rather a dynamic equilibrium exists between freely suspended bacteria and those adhering to particles. From the moment a bacterial biofilm forms, a detachment of cells or cell-aggregates takes place [44], dependent on the prevailing conditions. Several bacteria species, dependent on their nutrient supply, can exist either freely suspended or mainly aggregated in both pure and mixed cultures [45]. 2.1.4 Activated Sludge
Existing literature regarding protozoa and wastewater treatment deals mainly with aerobic processes, with the focus on activated sludge technology. This is due to the significance of this technology for wastewater treatment on the one hand and that suspended activated sludge is more easily accessible for biological investigations than slime-growth areas of fixed-bed reactors on the other. Activated sludge processes operate with typical sludge concentrations between 2–3 g l–1 [46]. About 70% of the activated sludge is organic content and 30% inorganic (clay: Si; Al; Fe; ferric oxide; calcium phosphate) [47]. Non- – or not easily – oxidizable organic matter makes up 20–25% of the sludge [41]. In a conventional activated sludge tank flocculate suspended material contains about 6 ¥ 109 bacteria ml–1, i.e., 1–3 ¥ 1012 bacteria g–1 dry weight [48]. They represent about 90% of the total biomass of the activated sludge. The proportion of living or metabolically active bacteria found in the flocs varies considerably, depending on the method of analysis. Estimates based on glucose, stearate and acetate uptake rates imply active proportions of 8–13%, 14–28%, and 5–10% of the total biomass, respectively [48]. More recently, direct measurements by fluorescence-microscopy indicate a proportion of 35–40% (de-
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hydrogenase activity [49]) and 70% (rRNA directed oligonucleotide probes, [50]), whereby a similar level of activity was assumed for all zones of the floc [51]. 2.2 Protozoa in Biological Wastewater Treatment Plants 2.2.1 Occurrence
Systematic investigations at a large number of wastewater treatment plants reveal protozoa as typical components of the biocoenosis (Table 3). Thus, for example, in all ten South African activated sludge plants studied by Bux and Kasan [52] “basic communities” of protozoa, typical for sewage plants were found. Similarly, Curds and Cockburn [53] found protozoa biocoenoses in 53 of 56 British activated sludge plants and all 52 biological percolation filter plants studied. In New Jersey, Chung and Strom [54] found protozoa in all the rotating disc contactors and according to Madoni and Ghetti [55], typical ciliate communities were detected in 38 of 39 activated sludge plants and 47 of 49 rotating disc contactors in the Emilia region of Italy. The presence of protozoa is closely associated with biofilms and restricted mainly to aerobic processes and therefore to certain areas of the wastewater treatment plant; only a few specialists among the protozoa take part in anaerobic processes. Thus protozoan communities can be typically encountered in activated sludge tanks as well as in the sedimentation tanks, whereas no protozoa are found in sludge digestion or in the supernatant of the sedimentation tank (effluent), with the exception of malfunctions [56].
Table 3. A survey of the protozoan fauna in sewage treatment plants (only microfaunistic in-
vestigations based on ten and more plants are taken into consideration), according to [52–55] Type of plant
Activated sludge
No. of plants investigated (country)
56 (Great Britain) 39 (Italy) 10 (South Africa) Trickling filter 52 (Great Britain) Rotating biological 49 (Italy) contactor 10 (USA)
a b
Occurrence of typical protozoan communities
Typical protozoan communities absent
Protozoa absent
Within 53 plants Within 38 plants b Within all 10 plants Within all 52 plants Within 47 plants b
2 plants a 1 plant b – – 2 plants b
1 plant ? – – ?
Within all 10 plants –
–
No ciliates, but flagellates present. Only ciliates investigated, no comments on other protozoan groups such as flagellates and amoebae.
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2.2.2 Species Composition
The majority of microfaunal investigations confirms that all of the three main groups of protozoa – flagellates, ciliates, and amoebae (naked and shell) – can be found in wastewater treatment plants, whereby ciliates form the largest proportion with regard to biomass and number of species, both in activated sludge [53, 57–62] and in fixed-bed processes (percolation filters: [53, 59]; rotating disc contactors: [63–65]), compare Table 4. It should be noted, however, that the composition of the protozoan biocoenosis, as well as that of the total biomass involved in the purification process, is mainly dependent on the composition of the wastewater, together with physical conditions and factors arising from the process technology used. In the case of malfunctions, or in the initial stage of a plant, very different compositions can be encountered. Sydenham [57] observed 2 municipal activated sludge plants over a period of 12 months and identified amoebae as the dominant group with regard to biomass. In sludge with a high organic load, Curds and Cockburn [66] and Mudrack and Kunst [67] report high population densities of flagellates. The age of the sludge also has an effect on the composition of the protozoan community. Kinner and Curds [63] quote 6–12 months as the length of time required to establish a steady-state community of protozoa in a pilot rotating disc contactor plant supplied with domestic effluent. Bacteria were visible on the disc surfaces within one day of startup followed within a few days by flagellates and small amoebae. Free-swimming bacterivorous ciliates appeared within 8–10 days. Subsequently, sessile peritrichous forms accompanied by carnivorous ciliates, rotatoria, and large amoebae make up the stable community. Parallel to sludge aging, a typical chronological succession of dominant protozoa populations can also be observed in activated sludge plants. After the initial phase of 1–2 weeks where flagellates, naked amoebae, and free-swim-
Table 4. Structure of the protozoan community in three urban activated-sludge plants, oper-
ating at different organic loading rates and dissolved oxygen concentrations (observation over a one year period), according to [62]. Biomass calculation is based on data, given by [61]
Organic load a O2-conc. (mg O2/l) Densities and biomass Ciliates Flagellates (< 20 mm) Naked Amoebae (
