Download Protozoa in Wastewater Treatment: Function and Importance

CHAPTER 3
Protozoa in Wastewater Treatment:
Function and Importance
Wilfried Pauli 1, Kurt Jax 2, Sandra Berger 1
1
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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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Protozoa in Wastewater Treatment . . . . . . . . . . . . . . . . . . 212
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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Impairments of Protozoa: Consequences for Water Purification . 241
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Environmental Biotechnological Aspects . . . . . . . . . . . . . . 243
4.1
4.2
Biodegradation Potentials of Ciliates . . . . . . . . . . . . . . . . . 243
Ciliates as Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . 245
5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
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
Protozoa in Wastewater Treatment: Function and Importance
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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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Protozoa in Wastewater Treatment: Function and Importance
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
Protozoa in Wastewater Treatment: Function and Importance
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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 <5 ¥ 106 – 5 ¥ 108 bacteria ml–1 – a concentration normally found only in more productive systems.” Large ciliates
(> 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
Protozoa in Wastewater Treatment: Function and Importance
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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)
Protozoa in Wastewater Treatment: Function and Importance
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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.
Protozoa in Wastewater Treatment: Function and Importance
215
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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W. Pauli et al.
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-
217
Protozoa in Wastewater Treatment: Function and Importance
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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W. Pauli et al.
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 (<50 mm)
a
kg BOD5/(kg MLVSS) day.
Plant 1
Plant 2
Plant 3
0.23–0.38
3.6–5.2
ind./ml mg/l
3000– 18–43
7400
43 000– 2.2–5.2
600 000
4000–
0.21–5.3
100 000
0.21–0.35
1.8–3.0
ind./ml mg/l
8600–
50–99
17 000
89000– 4.6–51
980 000
800–
0.04–
130 000 6.9
0.5–0.8
1.0–1.3
ind./ml
4500–
16000
38000–
1 600 000
77000–
101 000
mg/l
26–93
20–83
4.1–5.4
Protozoa in Wastewater Treatment: Function and Importance
219
ming ciliates predominate, more and more crawling and sessile forms appear,
which remain dominant throughout the stabilization phase and can be regarded as typical representatives of mature sludge [62, 65, 68–70]; see also Fig. 5.
Unlike the free-swimming forms, which arrive at the plant with the sewage and
are flushed out at the end of the process, the existence of sessile and crawling
forms is closely associated with the development of slime growth or sludge
Fig. 5. Composition of the bacterivorous ciliate community during the establishment of a
mature sludge. Stabilization, i.e., steady-state occurs after about 50 (activated sludge, above figure) and ca. 80 days (RBC, below figure), respectively. Bars lower than 100% indicate the additional presence of carnivorous and omnivorous ciliates, after [65]
220
W. Pauli et al.
flocs. Bound to biofilms as fixed slime growths (fixed-bed) or as sedimentable
sludge, they are retained in the treatment plant and can thus build up a stable
community with the bacterial flora. Whereas characteristic population succession takes place in both plant types, in percolation filters, due to the unequal
distribution of the organic load, a physical separation of the organisms is observed, dependent on the filter depth [68].
Figure 5a, b shows results from studies on the colonization behavior of ciliates in a pilot rotating disc contactor plant as well as in an operational activated
sludge plant [65]. Both plants were fed with domestic wastewater. Whereas in
the initial stage of the activated sludge plant ciliates make up between 0.17%
and 0.44% of the total biomass, in the stabilizing phase they account for more
than 9% of the sludge biomass. In the initial phase free-swimming forms from
the wastewater dominate. After 10–15 days their numbers drop markedly and
crawling (Aspidisca cicada, A. lynceus, Euplotes affinis, Chilodonella uncinata)
as well as sessile (Vorticella convallaria, V. microstoma, Epistylis plicatilis,
Opercularia coarctata) ciliates characterize the protozoan fauna. Similarly, in
the rotating disc contactor plant, ciliates makes up only 4–5% of the slime biomass in the colonization phase, as opposed to 12–19% under steady-state conditions. Here, too, essential changes take place during the colonization of the
submerged contact aerator and the typical ciliate biocoenosis develops in the
plant itself. In the initial phase, free-swimming ciliates such as Paramecium
putrinum and Uronema nigricans are present; in the stable phase sessile forms
such as Opercularia coarctata and Vorticella convallaria dominate. Investigations by Madoni [64, 65] make it clear that in both types of plants (submerged
contact aerator and activated sludge) a significant positive correlation exists
between the increase of the sludge, biofilm and ciliate biomass (r2 = 0.927 and
r2 = 0.853). This implies a close relationship between the size of the ciliate
population and the bacterial biomass.
2.2.3
Plant Specific Basic Communities
The relative abundance of an organism in a particular habitat can be considered as a measure for its significance within the ecological structure of the biological system concerned. Alongside amoebae and flagellates, Curds and
Cockburn [53] identified 67 and 53 ciliate species in 56 activated sludge plants
and 52 percolation filter plants in Britain, respectively. Madoni and Ghetti [55]
detected 45 and 47 ciliate species in 39 activated sludge plants and 49 percolation filter plants in Northern Italy. Of note is that the British and Italian activated sludge plants revealed very similar ciliate fauna [55]. Nevertheless, not all
species in the individual samples can be regarded as typical, as to their presence
and population density, for the respective wastewater treatment process. The
majority of the species are found only sporadically in a few samples and usually
with a low population density. The overall picture of the ciliate population is determined by a few, primarily sessile (peritrichous) and crawling (hypotrichous),
species most of which are bacterivorous (compare with Fig. 6). With cell counts
of, on average, more than 104 ml–1, ciliate densities are 100–1000 times higher
Protozoa in Wastewater Treatment: Function and Importance
221
Fig. 6. Examples of free swimming (holotrichous), crawling (hypotrichous), and sessile (peritrichous) ciliates in waste water treatment plants
here, than in the plankton of oligotrophic (10 ml–1) and eutrophic (100 ml–1)
waters [24]. Table 5 summarizes the dominant ciliate species in the “basis community” of each plant type identified by Curds and Cockburn [53] and Madoni
and Ghetti [55]. The specific biocoenosis differs according to plant type and to
the current operating conditions [55]: in areas with a high organic load an increase in free swimming species is observed [61, 66] along with a decrease in the
diversity of species [71, 72]; with these limitations, the community forms given
in Table 5 can be considered average for municipal plants.
2.2.4
Biomass
In activated sludge plants a high proportion of the eukaryotic biomass is comprised of protozoa. Investigations carried out by Sydenham [57] revealed that
protozoa made up over 90% of the total eukaryotic biomass of two municipal
wastewater treatment plants. According to Aescht and Foissner [61], protozoa
made up 99–100% of the eukaryotes in a pharmaceutical plant with a bacterial
nutrient load. The average proportion of protozoa in relation to total solids
(dw) is 5% [59, 73]. Ciliates alone make up 10% of the total biomass (pro- and
eukaryotic dry weight). Even higher numbers of ciliates are encountered in municipal rotating disc contactors where proportions of about 20% of the total
biomass of the slime-growth can be observed [64, 65].
2.2.5
Ecological Framework
The biocoenosis in wastewater treatment plants should not be regarded as a
community with a rigid composition and constant characteristics but rather as
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W. Pauli et al.
Table 5. Ciliate species dominating and occurring with a high frequency in sludge samples of
British (GB) and North Italian (I) sewage treatment plants, respectively, after [53, 55].
“Dominating” refers to the relative cell density, whereas “present” indicates the number of
samples, in which the respective species – independent of its individual numbers – could be
observed
Dominant Present
(%)
(%)
GB I
GB I
Life form
Ecological type
Nutrition a
Activated sludge (GB and I)
Aspidisca costata a
Vorticella convallaria a
Trachelophyllum pusillum a
Opercularia coarctata a
Carchesium polypinum a
Vorticella alba (GB)
Vorticella microstoma (GB)
Euplotes moebiusi (GB)
Vorticella fromenteli (GB)
Euplotes affinis (I)
Zoothamnium pygmaeum (I)
Trochilia minuta (I)
35
19
15
12
11
11
10
5
4
–
–
2
Crawling
Sessile
Free swimming
Sessile
Sessile
Sessile
Sessile
Crawling
Sessile
Crawling
Sessile
Crawling
Bacterivorous
Bacterivorous
Carnivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Filamentous b
Trickling filter (GB)
Opercularia micodiscum
Carchesium polypinum
Vorticella convallaria
Chilodonella uncinata
Opercularia phryganeae
Opercularia coarctata
Vorticella striata
Aspidisca costata
Cinetochilum margaritaceum
44
15
10
4
4
2
2
–
–
81
62
83
90
90
56
52
56
54
Sessile
Sessile
Sessile
Crawling
Sessile
Sessile
Sessile
Crawling
Crawling
Bacterivorous
Bacterivorous
Bacterivorous
Filamentous b
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Rotating biological contactor (I)
Euplotes moebiusi
Paramecium caudatum
Trachelophyllum pusillum
Vorticella convallaria
Opercularia microdiscum
Opercularia coarctata
Paramecium trichium
Cinetochilum margaritaceum
Chilodonella cucullulus
53
46
41
53
41
33
27
23
18
79
79
59
57
45
37
43
37
41
Crawling
Free swimming
Free swimming
Sessile
Sessile
Sessile
Free swimming
Crawling
Crawling
Bacterivorous
Bacterivorous
Carnivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Bacterivorous
Filamentous b
a
85
77
30
23
26
–
10
5
–
59
33
23
69
58
64
54
25
38
75
35
31
11
–
12
90
84
58
25
28
–
10
7
–
69
33
25
Dominant both in British and Italian plants.
Filamentous: ciliates with a specialized oral apparatus, enabling the ingestion of rod-shaped, filamentous bacteria.
– not present.
b
Protozoa in Wastewater Treatment: Function and Importance
223
an artificial but biological segment of natural self-purification processes, the
composition of which is influenced by ecological conditions and physico-chemical factors, thus differing from plant to plant and even within a plant over
time.
2.2.5.1
Sludge Loading
Sludge loads with fm-values between 0.2 and 0.6 [g BOD (g MLSS · day)–1] are
considered optimal for the purification sequence at conventional municipal activated sludge plants (e.g., [47, 67]). Ciliate densities of 6000–30,000 ml–1 are
found in sludge with these loads [71, 74]. However, similar concentrations of
ciliates are also encountered in sludge with both higher and lower loads:
Salvado and Gracia [71] observed a constant ciliate population density in a municipal plant with fm-values varying from 0.03 to 0.4. Experiments by Lee et al.
[74] confirm only slight changes in ciliate counts at sludge loadings between
0.1–1.4 [g BOD (g MLVSS day)–1]. Only under very heavy loads [1.8–2.4 g BOD
(g MLVSS day)–1], was a reduction in cell density observed.
Although the population density remains constant over a wide range, the
organic load influences the number of species and the composition of dominant ciliates in the basis community. The number of species present sinks with
increasing organic content of the wastewater [66, 71, 72]. According to Curds
and Cockburn [66], activated sludge with a relatively low organic load
[fm = 0.1–0.3 g BOD (g MLSS day)–1] shows the greatest species diversification,
whereby all three groups of ciliates – peritrichs (sessile), hypotrichs (crawling),
and holotrichs (free swimming) – are represented with approximately the same
number of species. In the medium load range of fm = 0.3–0.6, peritrichous species dominate and by high organic loads of fm = 0.6–0.9 equal portions of peritrichs and holotrichs are present (Fig. 7).
2.2.5.2
Temperature
Temperatures in municipal plants are generally slightly above the outside temperature in winter and slightly below in summer. Performance is optimal between 10 °C and 25 °C [41]. No negative effects on ciliate fauna are found up to
30 °C; experimental activated sludge investigations reveal a decline in ciliates at
temperatures above 30 °C and their disappearance above 40 °C [74]. The authors
discuss the concomitant deterioration of the settling properties of the sludge as
possibly resulting from the collapse of the ciliate population.
2.2.5.3
pH-Value
Activated sludge has a relatively high buffer capacity. If no strongly acidic or alkaline effluents are introduced, mainly from industrial processes, pH-values
generally fluctuate between 6.5 and 8 [41, 67, 75]. Therefore not only the tem-
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W. Pauli et al.
Fig. 7. Composition and species number of ciliates in activated sludge plants operated at different sludge loadings [food to micro-organism (F/M) ratio]. Results from an investigation of
52 British plants made by Curds and Cockburn [53]. Peritrichous, hypotrichous, and holotrichous ciliates represent sessile, crawling, and free swimming ciliates, respectively. In conventional municipal plants, treating domestic wastewater, a sludge loading between 0.2 and
0.6 is regarded to be optimum for the functioning of the sewage treatment process
perature, but also the pH-values of municipal plants are in a favorable range for
protozoan growth [75].
2.2.5.4
O2-Content
Conventional processes of biological wastewater treatment utilize the metabolism of the organic load, which is faster, more thorough, and easier to control
under aerobic conditions. Aerobic conditions are also a prerequisite for a high
incidence of protozoa. Few specialists can survive strictly anaerobic conditions
and little knowledge is available regarding their distribution or function in anaerobic degradation processes. The number of facultative anaerobic protozoa is
slightly higher, but almost all species seem to be able to survive low oxygen concentrations or even the absence of oxygen, at least for a short period [75]. Apart
from plant malfunctions (e.g., breakdown of the aeration), this ability is also
important in the normal cycle of activated sludge processes, where the organisms are constantly alternating between the aerobic activated sludge tanks and
the sedimentation tanks, in which anaerobic conditions arise for short periods
Protozoa in Wastewater Treatment: Function and Importance
225
in the deeper layers of the settling sludge (less than 4 h [41]). Only longer and
repeated oxygen deprivation over several hours (continual alternation between
6 h aerated and 24 h without aeration) leads to a marked decline of the sessile ciliates Vorticella convallaria and Opercularia coarctata, typically found in
wastewater treatment plants [76].
2.3
Significance of Protozoa for Wastewater Treatment
As already described (Sect. 2.2.2), the majority of protozoa in aerobic biological
purification systems are sessile or crawling ciliates. Whereas free-swimming ciliates are flushed out with the clarified water, crawling and especially sessile
forms are bound to bacterial biofilms (flocs and slime growths) [59]. In the case
of fixed-bed plants they remain bound to the biofilms in the plant; in activated
sludge processes they sediment with the sludge and are retained in the plant
due to continual sludge recycling.
To understand the role of protozoa and classify their position in the artificial
system of biological wastewater treatment, the following characteristics have to
be considered: type of motion (free swimming, crawling, or sessile); form of
nutrition (e.g., filter-feeders, browsers); sources of nutrition (abiotic colloids
and particles, bacteria, algae, other protozoa). From their form of nutrition and
their trophic level, functional aspects important for wastewater treatment become apparent. New understanding of natural systems as well as experimental
results on the physiology, energy budget, and nutrient cycling of both aquatic
and terrestrial protozoa provide extensive information regarding the ecological
role of this group of organisms, which, although quantitatively less significant
than bacteria, make a considerable contribution to wastewater treatment.
2.3.1
Nutrition
Several possibilities are open to ciliates for nutrient-uptake. On the one hand,
similar to bacteria, substances can be transferred directly through the plasma
membrane into the interior of the cell. Active and passive, carrier-mediated
uptake mechanisms through the plasma membrane have been described for
Tetrahymena for amino-acids [77–79], di-peptides [80], acetate, glucose [81,
82], and even for such complex nutrient solutions as proteose-peptone-yeast extract (PPY) medium [83]. Another method of nutrient uptake is pinocytosis
[84, 85]. It describes the active transport of dissolved substances in sub-microscopic, particle-free vacuoles or vesicles from the plasma membrane to the cell
interior, where they undergo normal lysosomal digestion processes. Finally,
ciliates have a highly specialized oral apparatus for taking up particulate matter by phagocytosis. The particles are not simply ingested with the surrounding
solution but rather undergo a highly efficient filtration process, facilitating the
concentration of particulate matter from a large volume of liquid, prior to their
intake in food vacuoles [85]. This process involves the production of a water
current by cilia (Fig. 8) and the extraction of particles from the flowing water
226
W. Pauli et al.
Fig. 8. Mechanisms of filter-feeding (ambiguously often referred to as ‘grazing’) used by protozoa. Water currents are created by flagella or the coordinated activity of cilia, that bring suspended food to the mouth region of the cell
with the aid of a ciliary sieve, which retains – in the case of bacterivorous species – particles sized between 0.3 mm and 5 mm [85–87]. The particles, thus concentrated, are subsequently ingested. Apart from food, abiotic and even indigestible matter of the size of bacteria are efficiently ingested [86–89]. Paramecia
concentrate food particles in this manner in their oral cavity up to 1000-fold
[90]. A similarly high concentration capacity can be assumed for Tetrahymena:
Whereas a volume of 50–80 nl is cleared of particles per hour and cell [87, 91],
a more than 1000 times lower water volume of 36 pl h–1 and cell is actually ingested by the food vacuoles [92]. The efficiency of this form of nutrition is underlined by investigations comparing the growth kinetics of Tetrahymena pyriformis with particulate and dissolved substances as nutrient source, respectively [93]. While under monoxenic conditions with particulate bacterial
substrate the half maximum growth rate is already attained with a bacteria content of 12 mg l–1 Klebsiella aerogenes (5.5 mg carbon l–1), 200 times that concentration of organic matter is required in case of dissolved nutrients (2.4 g l–1
proteose-peptone-yeast medium = 1.3 g carbon l–1).
Protozoa in Wastewater Treatment: Function and Importance
227
2.3.2
Reduction and Elimination of Suspended Particles and Bacteria
2.3.2.1
Clearing Rate
The volume of water cleared per individual and hour depends on cell size. Small
protozoa with cell diameters of less than 5 mm, such as flagellates, filter less than
1 nl h–1 at temperatures between 9°C and 17°C [20]. Higher filtration rates are
observed for larger ciliates. Sanders et al. [20] quote a yearly fluctuation range
of 12–156 nl h–1 for the filtration performance of planktonic ciliates. In laboratory experiments with the ciliates Halteria grandinella (diameter: 25 mm) and
Strombidium sp. (size: 15 ¥ 21 mm) filtration rates of 80–90 nl h–1 at 9°C and
120–140 nl h–1 at 17°C were determined. In the case of Vorticella microstoma
(average cell dimensions: 60 ¥ 30 mm), a ciliate frequently present in wastewater treatment plants, filtration rates as high as 156 nl h–1 at bacteria densities of
106 ml–1 are reported. Tetrahymena (cell dimensions: 40 ¥ 20 mm), a species
present but not dominant in wastewater treatment plants, has a filtration performance of 80 nl h–1 [91]. Fenchel [87] observed filtration rates of 50 nl h–1 and
cell at 20–22°C for Tetrahymena pyriformis and 200–1000 nl h–1 for larger
(100–200 mm) representatives of crawling and free-swimming ciliates such
as Euplotes, Paramecium, or Blepharisma. Assuming average filtration rates of
100 nl h–1 and cell and ciliate densities of 10,000 ml–1 and above [61, 94–97], this
implies that the entire liquid of an activated sludge plant can be filtered in less
than 1 h. The enormous predator and selection pressure exerted on the bacteria
is illustrated by the following examples.
Many heterotrophic bacteria in activated sludge have the ability to divide
every 20–40 min under optimal laboratory conditions [41, 48]. Under “field”
conditions, such as those prevailing in wastewater treatment plants, their
growth is generally much slower due to sub-optimal physical (temperature)
and physiological (nutrients, pH-values) parameters. The actual bacterial division rates under constant operating conditions and good nutrient availability
can be estimated from the ratio of the surplus (drawn off) sludge to the total
sludge in the activated sludge plant [98]. For low to high organic loads
(fm = 0.05–0.6 g BOD per g MLSS and day), growth rates can vary from 4–50%
per day [41] or, expressed in other terms, the bacteria population in the sludge
doubles every 48 h at most, i.e., in a time span by no means adequate to compensate for potential protozoan feeding.
Highly loaded wastewater contains ca. 106 bacteria ml–1. The majority are
medically harmless but others are pathogenic and bear health risks. Conventional wastewater purification involves an initial pre-clarification step of
20–30 min, after which the wastewater is fed into the activated sludge tank and
aerated for 4 h. In the aerated and agitated system of the activated sludge tank
the wastewater is brought into contact with a mixed microbial population in the
form of a flocculent suspension. When the desired degree of treatment has been
achieved, the flocculent microbial mass, known as the “sludge”, is separated for
2–4 h from the treated wastewater in a separate, specifically designed sedimen-
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tation tank. The supernatant from the separation stage is the treated wastewater, and should be virtually free of sludge. Most of the settled sludge from the
separation stage is returned to the aeration stage to maintain the sludge concentration in the aeration tank at the level needed for effective treatment and to
act as a microbial inoculum. Some of the sludge is removed for disposal, and is
known as “waste” or “surplus” sludge. In both the activated sludge and the sedimentation tanks, the resident ciliate community has sufficient time to filter the
entire wastewater several times, thus removing bacteria and abiotic particles of
similar size (see Sects. 2.3.1 and 2.3.2).
2.3.2.2
Experimental Findings
It has long been known that protozoa are present in wastewater treatment
plants and that their species composition reflects the prevailing conditions in
the plant. However, scientific opinion was less unanimous with regard to the actual contribution of protozoa to the purification process. Although Ardern and
Lockett [99], Pillay and Subrahmanyan [100], Pillay et al. [101] and McKinney
and Gram [102] referred to a connection between protozoa and the quality of
the water discharged from the plant, proof of a causal relationship was lacking
or inconclusive.
Curds et al. [103] succeeded in selectively removing protozoa from activated
sludge and further cultivating this protozoan-free sludge in bench-scale treatment plants over a long period. Through the subsequent re-introduction of typical sludge ciliates they observed, under various starting conditions, positive
effects on a series of parameters describing the success of the purification process (Table 6). The principal observation of their experiments was that in the
absence of protozoa the effluent of the plant was turbid, due to its high content
of suspended bacteria; this turbidity almost disappears after re-introduction of
the protozoa (Fig. 9).
Similar findings are published by Sridhar and Pillai [104] and Macek [105] in
protozoan-free, pasteurized sludge and in bacteria cultures isolated from activated sludge. The addition of sessile, crawling, and free-swimming ciliates
Table 6. Effects of ciliated protozoa on the effluent quality of bench-scale activated-sludge
plants. Results are given in mg l–1 unless otherwise noted; after [103]
Effluent analysis
Without ciliates
With ciliates
Mean reduction
BOD
COD
Permanganate value (4 h)
BOD after filtration
COD after filtration
Organic nitrogen
Suspended solids
Optical density at 620 nm
Viable bacteria counts (106 ml–1)
53–70
198–250
83–106
30–35
31–50
14–21
86–118
0.95–1.42
160
7–24
134–142
62–70
3–9
14–25
7–10
26–34
0.23–0.34
1–9
75%
38%
30%
81%
39%
51%
71%
76%
97%
Protozoa in Wastewater Treatment: Function and Importance
229
bacterial density
Fig. 9. Influence of ciliates on the bacteria content in the effluent of a bench-scale activated
sludge plant, after [103]
(Epistylis articulata, Vorticella microstoma, Aspidisca cicada, Chilodonella uncinata, Stylonychia putrina, Colpidium camylum) reduces high COD values and
suspended matter content. Farrah et al. [106] confirm the causal relationship
between the presence of ciliates and a clear, almost bacteria-free effluent with a
low organic content. Departing from a typical pro- and eukaryotic sludge biocoenosis, the authors show that a largely selective reduction of protozoa, by the
addition of sodium fluoride (0.2 mol l–1) or sodium azide (6–20 mmol l–1) results in a notably higher content of freely suspended bacteria including streptococci. After application of the eukaryotic cell toxins, the total count of fecal
streptococci increases about threefold and the proportion of suspended bacteria, as compared to those bound to flocs, increases from 0.3% to 64%.
Kakiichi et al. [107] made essentially the same observations. The effects of two
amphoteric detergents (orthodichlorobenzene and polyhexamethylene biguanide hydrochloride) with known effects on bacteria and protozoa were studied
and a causal relationship between poor quality of the outflow (increased turbidity and COD values) from batch cultures of activated sludge and the inhibitory
effect (reduction in population density) on the protozoa was observed. A correlation between the effluent quality and the population density of protozoa is
also implied by Lee et al. [74]. Studies with a bench-scale activated sludge plant
(organic load: 0.1–0.4 g BOD per g MLSS and day) show that the selective decline of the ciliate population density, due to running temperatures of 36°C and
over (see Sect. 2.2.5.2), corresponds to a more than twofold increase in suspended matter in the effluent.
Experiments with bacteria-free synthetic wastewater (e.g., [103]) exhibit that
freely suspended bacteria, originating from the autochthonous microflora of
the activated sludge itself, are substantially reduced in the presence of protozoa.
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W. Pauli et al.
Furthermore Curds and Fey [108] observed that bacteria originating from the
influent wastewater are also effectively removed in the presence of protozoa.
After mechanical destruction of the protozoan population, by means of a ball
mill, the authors determined concentrations of 6.5 ¥ 105 culturable E. coli ml–1
in the effluent of a continuously operating bench-scale activated sludge plant;
after re-inoculation of the activated sludge with ciliates (Opercularia coarctata,
Vorticella microstoma, Hypotrichidium conicum, Tetrahymena pyriformis) and
the establishment of a stable protozoan community, this count was reduced tenfold to 6.3 ¥ 104 ml–1. The half life of E. coli in the activated sludge was reduced
from 16 h to 1.8 h.
Filter-feeding ciliates in wastewater treatment plants are, in principal, not
selective consumers. Along with harmless bacteria, a series of pathogenic
strains causing, for example, diphtheria, cholera, typhoid, and streptococcal infections are also phagocytosed (for reviews [75, 109]. Investigations by Farrah
et al. [106], with activated sludge in batch cultures, illustrate the significance of
this elimination of pathogenic bacteria from wastewater treatment plants. After
selective reduction of the protozoan fauna by sodium fluoride (200 mmol l–1) or
sodium azide (20 mmol l–1), cultures of Salmonella typhimurium and E. coli, added in densities of 105 ml–1 almost treble within 24 h (S. typhimurium and sodium fluoride) or only decrease by ca. 50% (E. coli and sodium azide), whereas
in untreated controls with protozoa, both bacteria are reduced to less than 5%
of their initial density. Moreover, under conditions of aerobic sludge stabilization, the authors show that even low densities of protozoa (660 ml–1) lead to a
substantial elimination of bacteria. Figure 10 shows results with Streptococcus
faecalis.
Fig. 10. Effect of sodium azide (6 mmol l–1, selectively reducing protozoan activity) on
Streptococcus fecalis in activated sludge (laboratory scale), after [106]. CFU: colony forming
units
Protozoa in Wastewater Treatment: Function and Importance
231
2.3.2.3
“Field”-Observations
“Field” observations leave no doubt that the results found in the laboratory
microcosms are transferable to pilot and full-scale plants and that the presence
of a typical protozoan community is reflected by the improved quality of the
plant effluent.
First, a close negative correlation is observed between the population density
of, mainly crawling and sessile, ciliate populations and the proportion of suspended matter in the effluent of wastewater treatment plants (domestic and
municipal wastewater [56, 110–114] and brewery wastewater [115]). Results
from a three-year investigation of three activated sludge plants with different
organic loads in Spain [114] reveal – on average for all plants – a highly significant correlation coefficient between total ciliate population density and biological oxygen demand of r = – 0.868. In the presence of protozoa the effluent BOD
ranges from 4 mg l–1 to 18 mg l–1, rising to values of up to 67 mg l–1 in their absence. An almost identical correlation between effluent quality (COD) and the
population density of typical activated sludge ciliates was observed by Sudo and
Aiba [111] for six municipal wastewater treatment plants in Tokyo. Mean COD
values of 10 mg l–1 were found with ciliate densities of ca. 104 ml–1; these increase to 40 mg l–1 when ciliate densities drop to 102 ml–1 (Fig. 11).
Second, according to Curds and Cockburn [53], plants without ciliates can be
recognized by the low quality of their effluent: 3 out of 53 activated sludge
plants were selected due to the high content of suspended material in their
effluent. In one of these plants no protozoa could be found at all, in the other
two no ciliates, only small flagellates, could be detected. The highest BOD values
measured in the three plants occurred in the plant with no protozoa.
Fig. 11. Relationship between protozoan densities and effluent COD, observed in municipal
activated sludge plants of Tokyo, after [111]. Symbols represent different plants
232
W. Pauli et al.
Finally, as compared to normal activated sludge processes, the clarifying effect of protozoa in activated sludge processes with submerged fixed-bed filters
– a technology which creates additional surfaces for slime growth and primarily sessile ciliates [116–118] – improves, which is basically due to low bacteria
and suspended matter content in the fixed-bed plant effluent [117, 119].
(Evidently, protozoa find optimum living conditions on the filter installed in the
activated sludge tank, an adequate oxygen supply and plenty of food, so that the
dense population of mainly ciliates even crowds out attached bacterial growths.
In contrast to the common activated sludge process, where ciliates contribute to
about 10% of the total, bacteria dominated biomass, an almost inverse relation
of 68% protozoan and 32% bacterial biomass (dw) is found for the biofilms of
submerged fixed-bed filters [116].)
2.3.3
Elimination of Dissolved Substances
The bulk of dissolved substances entering the wastewater treatment plant are
amino-acids, products of protein hydrolysis in the sewage system, and fatty
acids. Carbohydrates are usually completely degraded in the sewage before
reaching the plant.
Although many protozoa can take up organic substances [85, 89, 120, 121],
their contribution to the degradation of these substances in wastewater treatment plants is negligible: For these substances the essential activity comes from
the bacteria population. They dominate the biomass and possess a higher metabolic efficiency as a result of their high surface to volume ratio [41, 46–48, 67].
An impression of the different degradation efficiencies can be gathered from
measurements of amino-acid uptake by Escherichia coli and T. pyriformis [122].
Even under the assumption that all ciliates present in wastewater treatment
plants can metabolize not only bacteria but also dissolved substances similar to
T. pyriformis, the experiments reveal an 80-times higher uptake of amino-acids
by bacteria. Results from Hrudey [123] can also be well interpreted in the light
of the significantly higher degradation rate of dissolved substances by bacteria.
After addition of peptone, a protein hydrolysate rich in amino-acids, an immediate rise in the bacterial biomass was observed, whereas ciliates were scarcely
able to convert the available peptone into their own biomass and could only reproduce substantially after the bacterial content increased considerably.
2.3.4
Flocculation and Composition of the Bacterial Community
Apart from the feeding activity of protozoa, another factor is discussed as contributing to the reduction of the content of suspended matter and bacteria in
bench and full-scale plants. In the presence of protozoa, freely suspended, single
bacteria form compact flocs, which then settle [59, 105, 106, 111, 124–128].
This is attributed, on the one hand, to polymer, particle-aggregating excretion products (polysaccharides) from protozoa [59, 125], which are possibly released into the media to facilitate a more effective uptake of particles [24, 129].
Protozoa in Wastewater Treatment: Function and Importance
233
On the other hand, this flocculation is believed to be associated with the
exocytosis of indigestible, originally finely dispersed material as a digested
bundle [130, 131], which in turn could serve as a settlement surface for solitary
bacteria [132–134]. However, wastewater itself contains a high proportion of
chemically complex particles of differing sizes, and bacteria themselves, dominant with regard to their biomass in wastewater treatment plants, produce extracellular polymeric substances (polysaccharides), to which they can effectively adsorb [135, 136]. For these reasons, protozoa, by excretion of digested remains and polymers, probably play only a minor role in floc formation in
wastewater treatment plants.
Bacteria feeding itself seems, not only quantitatively but also qualitatively, a
significant stimulus for complex bacterial growth forms. As a result of the predator-prey relationship between protozoa and bacteria, a collapse of the bacteria population in the activated sludge and a reduced elimination efficiency of
the system as a whole would be expected (see Sects. 2.1.2 and 2.3.3). Such
collapses or phase-shifted oscillations between predator and prey can be observed in model systems [111, 137–141] and in natural ecosystems [20, 142–146]
and led originally to the view that protozoa are harmful for the clarification
process [147].
Only a few protozoa, e.g., amoebae, mostly present at low densities in wastewater treatment plants, are principally capable of taking up larger particles, due
to their ability to entrap their prey. Ciliates, typical representatives of protozoa
found in wastewater treatment processes, possess a highly specialized oral apparatus for highly efficient filtration, which at the same time exclude particles
of several micrometers in diameter [86]. The ability of bacteria to develop larger forms, to grow collectively, or to merge as micro-colonies protects them
against the predator pressure from the protozoa [148–153]. The development of
growth forms resistant to filter-feeding can thus be seen as an essential process
in the evolution of bacterial flocs and biofilms [45, 111, 127, 148].
To what extent a qualitative selection of floc and biofilm forming bacteria is
possible [148], and what could be gained from a quantitative shift within a species to larger or aggregating phenotypes [45, 149], cannot be decided in the light
of the present literature. Güde [148] observed selection of bacteria populations
which aggregate in pilot wastewater treatment plants. On the other hand
Shikano et al. [149] find that, in the presence of the ciliate Cyclidium sp., phenotypes of considerably larger dimensions appear within a bacteria species.
Gurijala and Alexander [154] provide evidence of lower feeding pressure by the
ciliate Tetrahymena thermophila on bacteria with hydrophobic surfaces – in
other words on phenotypes with water-repellent properties – which enhance
their adhesive, i.e., aggregation, ability [155].
Many bacteria are also capable of organizing themselves spontaneously into
biofilms in the absence of protozoa, thus forming flocs [102, 135, 156].
Nevertheless, the extent and persistence of the flocs seem to be influenced by
the presence of protozoa. Farrah et al. [106] show that in the absence of protozoa autochthonous aerobic bacteria and cultures of Salmonella typhimurium
and E. coli introduced into the sewage sludge are predominantly freely suspended (43–68%). In the presence of protozoa, the proportion of freely suspended
234
W. Pauli et al.
bacteria drops significantly to 1–15%. The majority can now be found in or
adhering to flocs (85–99%); compare also Fig. 10.
Experiments in model wastewater treatment plants [105] show that different
ciliate species induce flocculation to different degrees. With the exception of the
crawling Aspidisca costata, which, even at low population densities, induces
good flocculation when added to protozoan free (pasteurized: 50°C, 5 min) sewage sludge, the tendency of bacteria to aggregate in the laboratory fermenter
varies considerably for free-swimming (Colpidium campylum), crawling
(Chilodonella uncinata, Stylonichiaputrina), and sessile (Vorticella microstoma)
forms, essentially independent of their population density.
Ciliates feed selectively, not only – as shown for Tetrahymena – with regard
to the physico-chemical surface structure of their prey [154], but also regarding
the size of the phagocytosed particles: This was shown by Fenchel [86] with filter-feeding ciliates, characteristic for the ciliate fauna in wastewater treatment
plants. Each ciliate species can only filter specific size ranges of food particles,
i.e., different ciliate species feed in their respective – sometimes distinct – niches (Fig. 12). Dependent on the selection mechanism, different effects on the
composition of the bacterial populations and the development of more or less
aggregated growth forms become apparent.
Fig. 12. Clearing rate (volume of water the organisms can clear of particles per unit time
at low particle concentrations, here in multiples of the ciliates own volume per h) for three
ciliate species as function of particle size, from [86]
235
Protozoa in Wastewater Treatment: Function and Importance
2.3.5
Reduction of the Total Biomass
In order not to exceed a sludge concentration favorable for the purification performance of the wastewater treatment plant, an amount equal to the daily production must continuously be drawn off. This excess sludge is subsequently
concentrated, digested, and drained and must finally be disposed of as a potentially pathogenic and frequently toxic waste product. Excess sludge is therefore
an economic factor, even within the wastewater treatment plant itself. A reduction in sludge production corresponds to savings in personnel, energy, and running costs.
Since the function of the sedimentation tank is merely to separate the biomass from the purified water, the effluent concentration must already be attained in the well-mixed activated sludge tank. The organisms therefore live in an
environment with low nutrient concentrations, resulting in slow growth [46,
67]. The average age of activated sludge (sludge residence time) for organically
burdened municipal sewage, where the main emphasis is on the elimination of
the carbon compounds, is 4 days [41, 46]. If nitrification is an objective, the
sludge residence time increases to 8–10 days [157]. This means that the sludge
biomass doubles after 4 days, at the earliest. Generation times in this range imply not only stationary growth for the majority of heterotrophic bacteria but
also sub-optimal, reduced growth rates for the ciliate fauna having generation
times of 5–15 h; see Table 7.
In principal, a lengthening of the food chain results in a reduction of the originally available energy. In every heterotrophic link, part of the assimilated food
is converted into biomass. The remaining carbon compounds are used as
energy source for metabolic processes. When the chain becomes longer, less
energy will remain locked into biomass. This means more carbon-mineralization and less biomass production.
Table 7. Doubling times of activated sludge and ciliates isolated from activated sludge plants
Activated sludge a
Aspidisca costata b
Aspidisca lynceus b
Vorticella microstoma b
Vorticella convallaria b
Carchesium polypinum b
Opercularia spec b
Epistylis plicatilis b
Colpidium campylum b
Tetrahymena pyriformis b
Paramecium caudatum b
a
b
[158].
[111].
Doubling time (h)
Temperature ( °C)
3.3–10
13.6
12.4
5.0
7.6
9.3
5.0
10.2
4.7
4.5
12.0
20
20
20
20
20
20
20
20
20
20
20
236
W. Pauli et al.
Protozoa assimilate about 85% of readily exploitable nutrients after uptake.
They are converted into individual biomass or respired for energy purposes. The
remaining 15% are eliminated as compact digestion bundles (exocytosis) or dissolved substances (excretion) [159]. Under optimal growth conditions, ca. 50% of
the nutrients taken up by protozoa are converted into individual biomass, which
corresponds to the metabolic efficiency of prokaryotes [160]. Different circumstances are encountered under inhibited or stationary growth conditions. Here
the emphasis is on basal metabolism, not growth: The metabolic performance is
reduced and energy consumption, as mineralized carbon in the form of CO2, increases [128, 161]. This diminished ability to utilize available nutrients for biomass production as a result of reduced growth rates is demonstrated by Ratsak et
al. [128] with Tetrahymena pyriformis. At a high growth rate (generation time of
5.5 h near the optimum of 3.4 h), 51% of phagocytosed bacterial biomass
(Pseudomonas fluorescens) are converted into ciliate biomass, whereas at a low
growth rate with a generation time of 17 h only 39% of the prey is converted into
predator biomass. At the same time the ratio of respired mineralized carbon to
that converted into cell biomass increases from 0.65 to 1.2.
In municipal activated sludge plants ciliates are present in densities of
104 ml–1 and over [61, 75, 94–97]. The number of bacteria required to maintain
this ciliate population can be estimated based on data from Macek [162]. Under
steady-state conditions (20°C) and generation times of 5 days, free-swimming
ciliates such as Colpidium campylum and sessile forms such as Vorticella microstoma at densities of 1.3 ¥ 104 ml–1 and 0.59 ¥ 104 ml–1 consume, over the 5-day
period, 2.5 ¥ 109 and 2.1 · 109 bacteria ml–1 (450 and 420 mg COD l–1), respectively. The bacterial content of sewage arriving at the plant is on average
106 ml–1. With flow-through times of 2 h or more in municipal activated sludge
plants [41], no more than 0.5 ¥ 106 bacteria are available per ml and hour for
the ciliates. Based on the findings of Macek [162], however, a typical ciliate density of 104 ml–1 would require more than 17 ¥ 106 bacteria ml–1 and hour (2–3
¥ 105 ml–1 in 5 days). Therefore, the suspended bacterial content in the influent
sewage cannot essentially contribute to the production of protozoan biomass.
To supply adequately the protozoan population a 30-times higher bacterial content in the influent would be required.
It is known that bacterivorous species are capable of effective filtration and
ingestion of abiotic particles with diameters of 0.3–5 mm [85–87; see also
Sect. 2.3.1] and exploiting them, if possible, for cell reproduction or to increase
individual biomass. Thus in bench-scale plants, the addition of emulsified lipids, which form suspended particulate fat droplets, leads to a rapid increase in
sessile ciliates, which can accumulate these lipids in their cytoplasm [123].
Similarly, Tetrahymena is able to convert particulate suspended skimmed-milk
for reproduction, thereby attaining high population densities [163, 164]. It is unclear however to what extent particulate abiotic organic materials (e.g., protein
rich colloids from feces) in municipal sewage are suitable, in terms of chemical
composition, size, and content, to be utilized in the biomass production of typical sewage plant protozoan fauna.
The composition of the ciliate community in wastewater treatment plants is
primarily made up of bacterivorous filter-feeding organisms which efficiently
Protozoa in Wastewater Treatment: Function and Importance
237
concentrate and ingest particulate matter the size of bacteria from the surrounding liquid (see Sects. 2.2.3 and 2.3.1). Bacteria occur both in activated
sludge and fixed-bed processes as complex, aggregated cell formations (flocs
and slime growth). Firmly embedded in these structures, they are protected
against their protozoan predators. However, there is a dynamic equilibrium between flocculation and de-flocculation (see Sects. 2.1.3 and 2.3.4) which, in the
presence of protozoa, shifts towards more complex micro-colonies and, in their
absence, leads to high concentrations of single suspended bacteria (see
Sects. 2.3.2 and 2.3.4). That ciliates indeed can exploit the micro-flora of the
sludge itself as their primary source of nutrition is confirmed by experiments
with sterile synthetic wastewaters, e.g., [103, 123]. Activated sludge with an almost exclusively bacterial biomass was supplied with sterile synthetic wastewater as nutrient source (see Sect. 2.3.3) and nonetheless, a typical protozoan
biocoenosis is developing.
The average sludge concentration at municipal plants is quoted as 2–3 g (dw)
l–1 [46], which corresponds to ca. 6 ¥ 109 bacteria ml–1 [48]. In conventional
plants this bacterial mass is reproduced in 4 or more days (sludge residence
time). Referring to data from Macek [162], typical ciliate populations in activated sludge consume 1.5–2.9 ¥ 109 bacteria ml–1. In other words, even at shorter
retention times in a plant aimed primarily at the elimination of carbon compounds, a considerable proportion (25–48%) of the bacteria can be phagocytosed by ciliates: This corresponds to a 10–19% reduction of the accumulated sludge, based on a mineralization of around 40% of the bacterial food [128].
Observations with submerged fixed-bed filters in activated sludge plants reveal
a similar picture with regard to the reduction of the accumulated sludge by protozoa. In the activated sludge tank (volume 756 m3) a contact aerator, whose
slime-growth makes up almost 18% of the biomass (dw) of the tank, leads to a
reduction of the BOD sludge accumulation of about 25% [117]. Such submerged fixed-beds are primarily colonized by protozoa whereby ciliates dominate [116, 117, 165, 166], comprising around 68% of the total biomass [116].
Based on these data, an additional biomass of 12% consisting exclusively of
ciliates (18% additional biomass, 68% of it ciliates) effects a sludge reduction
of 25%. A transfer of these results to conventional activated sludge plants
would mean that the autochthonous ciliate fauna, as the second link in the food
chain and representing 9% (dw) of the total biomass [64, 65], is in a position
to reduce sludge accumulation by 19%.
2.3.6
Influence of Protozoa on Bacterial Metabolism
A series of studies on degradation efficiency in bench-scale wastewater treatment
plants show that in the presence of protozoa – in spite of their antagonistic effects
as bacteria predators – the physiological performance of the bacteria is maintained or even increased: In bench-scale plants, ciliates show no effects on the nitrification bound to flocs [103, 126, 127]. The degradation of nitrilotriacetic acid by
bacteria is equally unaffected by the presence of ciliates; however, here a shift
from single suspended to complex aggregate growth forms is observed [126, 127].
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W. Pauli et al.
Clear indications of an increase in bacterial metabolic activity were found by
Curds et al. [103]: Under experimental conditions they observed, in the presence of protozoa, an increased degradation (BOD, COD) of the dissolved, nonfilterable portion of organic materials, attributed almost exclusively to the activated sludge flora (see Sect. 2.3.3 and Table 6). Findings by Wiggins and
Alexander [167] also imply a positive influence of protozoa on bacterial degradation processes with regard to the organic pollutants 2,4-dichlorophenol (2,4DCP) and 2,4-dichlorophenoxyacetic acid (2,4-D). Although protozoan feeding
reduced the mixed culture of freely suspended bacteria by more than one order
of magnitude – leading to delayed degradation compared to protozoa-free cultures – after 15 days the environmental chemicals 2,4-DCP and 2,4-D were mineralized in the presence of protozoa to 70% and 90%, respectively: Whereas in
cultures where the protozoa were inhibited by nystatin and cycloheximide, degradation of only 40% (2,4-DCP) and 10% (2,4-D) were observed over the same
period (Fig. 13).
In biocoenoses other than wastewater, i.e., in microcosms with pure and
mixed cultures of typical aquatic and terrestrial bacteria, an increased bacterial
metabolism in the presence of protozoa is observed. Various explanatory attempts emphasize the direct influence of the protozoan metabolic activity;
others attach more importance to bacteria feeding and its indirect consequences on the size and composition of bacteria populations and some correlate the
micro-currents, generated by the ciliates, with an improved food and oxygen
supply of the bacterial flocs or multi-layer biofilms. Protozoa are capable of metabolizing bacterial metabolic products such as acetic-acid, butyric acid, and
ethanol [77, 168] and could thus avert end-product inhibition [169]. On the
Fig. 13. Effects of protozoa on the mineralization of 0.1 mg l–1 2,4-dichlorophenol and 2,4-
dichlorophenoxyacetic acid (2,4-D) in sewage. Cycloheximide (250 mg l–1) and nystatin
(30 mg l–1) were used to suppress protozoa; from [167]
Protozoa in Wastewater Treatment: Function and Importance
239
other hand, protozoa release a series of organic substances such as amino-acids
[170] and “growth factors,” having chemical structures not characterized in detail [22, 109, 171–175], into the surrounding medium, leading to activation of
bacterial metabolism and growth. Furthermore, protozoa have the highest
excretion rate of inorganic phosphate and nitrogen, relative to biomass, within
the zooplankton [176]. In addition, in the presence of protozoa, an accelerated
bacterial phosphorus mineralization is observed [177]. This mutually advantageous interaction by nitrogen and phosphorus re-mineralization is emphasized
by many authors [22, 145, 177–183]. To what extent these additional organic and
inorganic substances introduced into the wastewater cycle play a part in wastewater treatment processes is controversial, but due to the composition of the
wastewater, rather unlikely [75, 184]. On the one hand, municipal sewage itself
is a complex nutrient solution with a heavy organic load; on the other hand,
nitrogen and phosphorus are present in excess in wastewater treatment plants,
in contrast to most limnic, marine, and terrestrial ecosystems (a BOD:N:P ratio of 100:5:1 is considered to be the optimal substrate composition of sewage
– compared to this nutrient balance, municipal sewage with average BOD:N:P
ratios of 100:17:5 [41] contains an excess of nitrogen and phosphorus).
However, in the case of commercial and industrial wastewaters with high carbon loading and comparatively low concentrations of nitrogen and phosphorus
(e.g., vegetable processing businesses, fiberboard works, paper and cardboard
factories, coking plants, as well as chemical and pharmaceutical industries [41,
185]) catalytic effects on bacterial metabolism by interactions with N and P set
free by protozoa are quite conceivable.
It is not self-evident that bacteria feeding and their subsequent reduction of
bacterial populations should have positive effects on bacterial metabolic turnover. A possible cause could be the qualitative shifting of the selection conditions for the bacteria and therefore the composition of mixed bacteria populations and their organizational forms. The success of a bacteria population is not
only dependent on its adaptation to the nutrients on offer but also on whether
it is edible for protozoa [148]. As discussed in Sect. 2.3.4, the selection of feeding-resistant bacterial growth forms can be viewed mainly as a result of phagocytic activity of protozoa: Freely suspended bacteria are succeeded by aggregated, sessile growth forms [136, 148, 186]. That this shift can be accompanied
by a simultaneous intensification of the microbial metabolic processes is shown
by studies on marine bacteria, which as adherent cells in biofilms (“marine
snow”) display faster growth (incorporation of thymidin into DNA [187]), an
increase in electron transport (reduction of tetrazolium salts to formazan
[188]), and higher hydrolytic activity [189], than as freely suspended single
cells.
2.3.7
Filamentous Bacteria and Protozoa
Filamentous bacteria are present in the bacterial flora of almost all activated
sludge. Due to their large surface area, they are well-equipped for the adsorption and metabolism of organic compounds. At low densities, they contribute to
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W. Pauli et al.
the stabilization of activated sludge flocs. However processing problems arise if
mass reproduction of filamentous bacteria occurs in the activated sludge tank.
The enlarged surface area of the flocs hinders the settling and thickening processes in the sedimentation tank which can, in extreme cases, due to the formation of light, fluffy, poorly settling flocs, result in the discharge of sludge into natural waters. This phenomenon, known as “bulking sludge,” used to be caused
by “high load bacteria” such as Sphaerotilus sp. or filamentous types 1863 and
0961. Today, however, many of the filamentous bacteria found in sewage treatment are adjusted to low carbon concentrations [low F/M ( food:micro-organism) bulking], e.g., types 0041, 0675, 0092, 1851, or Microthrix parvicella.
Experience shows that putrid wastewater, rich in H2S or with high carbohydrate
or short-chain organic acid content (i.e., wastewater from food processing, paper and textile industries), as well as low nitrogen, phosphorus, or oxygen con-
Fig. 14. Degeneration of bulking sludge (decrease of sludge volume index: SVI) in the aera-
tion tank of an activated sludge plant and in laboratory experiment by the filamentous predacious protozoan Trochilioides recta; from [194]
Protozoa in Wastewater Treatment: Function and Importance
241
tent, stimulates the development of bulking sludge. Various chemical (e.g., liming, chlorination, addition of H2O2 , iron salts, and nitrogen and phosphorus
compounds) and physical (e.g., increased oxygen supply) methods are implemented to combat bulking. Sometimes even operational conditions of plants
are altered (e.g., increasing the return-flow rate, by-passing the pre-clarification, aerobic, and anaerobic selectors) [67, 185].
In principal, autochthonous ciliates appear suited to counteract abundant
development of filamentous bacteria. However, only a few species capable of
taking up filamentous bacteria are present in activated sludge plants, e.g.,
Trithigmostoma cucullus (Chilodonella cucullus), Trochilioides recta, Trochilia
minuta, and Chilodonella uncinata. If these ciliates attain a high population
density, a pronounced decline in filamentous bacteria and degeneration of
bulking sludge is observed within a few days, both in bench-scale and operational plants [190–193]; see Fig. 14. Effective cell densities for Trochilioides sp. are
quoted as 1000 ml–1 [190] and for Trithigmostoma cucullus and Trochilioides
recta as 2000 ml–1 [193].
3
Impairments of Protozoa: Consequences for Water Purification
Ciliated protozoa are very numerous in all types of aerated biological treatment
systems (compare Sects. 2.2.3 and 2.2.4). They play an important role in the
purification process removing, through predation, the major part of dispersed
bacteria that cause highly turbid, i.e., low quality effluent. It has been generally
recognized that changes in the population density and community structure of
ciliates affect the food web of this artificial ecosystem, thus influencing the performance of plants. Excess influx of toxic wastes with detrimental effects on ciliates would prevent clarification, thereby severely threatening the degradation
process. A variety of chemicals can limit growth of ciliates. As with organisms
from other taxonomic, functional, and trophic levels, the toxicological effects
induced by organic and inorganic chemicals on ciliates vary widely, i.e., EC50values ranking from some mg l–1 to some g l–1 (reviewed by [194, 195]). Substances having toxic effects which diminish or even paralyze the purification
performance frequently find their way into wastewater treatment plants with
commercial and industrial wastewater. Risks are particularly great from metalfinishing works with electrochemical processes and wastewater from iron and
steel pickling plants, accumulator-charging stations, stereotype, photocopy,
photographic and printing works, dry-cleaning premises, industries producing
pesticides, herbicides, and disinfectants, as well as tanneries, leather goods
manufacturers and coking plants. In order to estimate the hazard potential and
to lay down maximal concentrations, in addition to bacterial tests, biological
tests with ciliates are indispensable to reflect potential risks of hazardous substances on the biological system of wastewater treatment as a whole.
Tests with typical wastewater protozoa have been carried out for a number of
toxic substances. Gracia et al. [196] observed effects of copper (sulfate) in concentrations of 1 mg Cu2+ l–1 on species diversity and population density – especially of the ciliates – of natural sludge samples. Madoni et al. [197] determined
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W. Pauli et al.
the 50% lethal effect concentrations of Cu<Hg<Cd<Pb<Cr<Zn (1 mg l–1 –
50 mg l–1) on various ciliates isolated from activated sludge, whereby the authors report differences in species sensitivities of up to two orders of magnitude, dependent on the heavy metal tested. Kakiichi et al. [107, 198–200] report
inhibitory effects of disinfectants and surfactants on typical activated sludge
ciliates. A comparison of the effect potential of 4 disinfectants towards the
wastewater bacteria Alcaligenes faecalis and the wastewater ciliate Colpoda aspera reveals an almost 10-fold higher sensitivity of the ciliates [200]. Higher
sensitivities of ciliates in comparison to bacteria were also found by Yoshioka et
al. [201] for 32 wastewater relevant environmental chemicals. Results from the
OECD activated sludge respiration test (RI Test, [202]) – considered as an indicator for acute effects of chemicals on heterotrophic bacterial flora – and
growth tests with Tetrahymena, a ciliate typical in polysaprobic surface waters,
but also found in activated sludge and submerged contactor plants [53, 55, 111,
112, 115, 203–209] were compared: 50% effect concentrations were, on average,
10 times lower with the ciliate test. Furthermore, certain substances proved
highly toxic in the Tetrahymena test, and showed only weak effects in the respiration test; out of a total of 32 substances, just 6 cases had a (toxic) effect potential of less than 100 mg l–1. The weak correlation of r2 = 0.17 confirms the discrepancy between the two tests (Fig. 15). Similar observations of a low correlation were made by Pauli and Berger [210]. Figure 16 illustrates toxic responses
of 4 ciliate species and standard tests with activated sludge towards industrial
chemicals (data taken from the International Uniform ChemicaL Information
Database, IUCLID, including toxic data of a wide variety of industrial chemi-
Fig. 15. Acute effects of chemicals on the bacterial flora of activated sludge (OECD
Respiration Inhibition Test) in comparison to those on the ciliate Tetrahymena pyriformis
(growth inhibition) and on fish (OECD lethality test with Oryzias latipes); after [202]
Protozoa in Wastewater Treatment: Function and Importance
243
Fig. 16. Comparison of results from standard activated sludge respiration tests and bioassays
with ciliates (data from IUCLID); from [210]
cals). Although a generally higher sensitivity of ciliates cannot be observed for
this data set, the random distribution of points around the bisector confirms
the dissimilarity of ciliate and activated sludge toxicities (r2 < 0.01, n = 35).
Evidently ciliates are not only sensitive to pollutant induced stress, but test
results reflect a series of additional toxic interactions, not represented by tests
with bacteria in activated sludge. That this different toxic profile is probably due
to the more complex cell-physiological – eukaryotic – organizational structure
of the ciliates is implied by QSAR studies for heterogeneous chemical classes
[211], which revealed a high correlation between the LC50 values found in the
widely accepted fish lethality test (r2 = 0.78) with Tetrahymena growth, but not
with bacteria test.
4
Environmental Biotechnological Aspects
4.1
Biodegradation Potentials of Ciliates
Although it is well known that ciliate grazing on bacteria fulfills important tasks
in the biological purification of sewage (compare Sects. 2.3.2.2 and 2.3.2.3) and
that a number of technical methods and plant operation parameters obviously
improve the purification efficiency by favoring ciliate growth (see Sects. 2.2.5,
2.3.2.2, and 2.3.2.3); only recently some pioneering attempts have been made to
specifically use ciliates in biodegradation processes.
Generally, large amounts of biosludge are formed in biological wastewater
treatment processes and the separation, dewatering, treatment and disposal of
this sludge represents major investment and operating costs. One of the poten-
244
W. Pauli et al.
tially useful assemblies for reducing the sludge yield is the two-stage cascade
used in many experiments for the study of ciliate-bacterial interactions, e.g.,
[140, 212–215]. The technique of a two-stage system enables one to manipulate
the artificial ecosystem of conventional treatment processes so that dispersed
bacteria are growing in the first part of the process and being consumed by protozoa in the last. Whereas in conventional treatment due to the growth of floc
or film forming bacteria most of the bacterial biomass is protected against predation (see Sect. 2.3.4), dispersed bacteria can be readily taken up and metabolized by protozoa (see Sect. 2.3.2), resulting in a lower sludge yield (see
Sect. 2.3.5). Operating the first part of the treatment process as an aerated tank
reactor without biomass retention and at an hydraulic retention time short
enough to prevent a significant growth of protozoa is a simple way to stimulate
this growth of dispersed bacteria. Cultivations using synthetic wastewater and
defined cultures of bacteria and ciliates in a two-stage chemostat cascade have
shown that protozoan grazing can result in a considerable biomass reduction
[128]. By introducing a “predation trap” (second stage) it was possible to obtain
a decrease of 12–43% in biomass yield in comparison with a system without ciliate grazing. Studies of Lee and Welander [216, 217] confirm this potential of a
two-stage system to reduce the sludge yield. Employing synthetic wastewater
and mixed cultures of bacteria, protozoa and metazoa from activated sludge
they observed a sludge yield around 30–50% of the yields typically obtained in
conventional aerobic processes [216]. If authentic instead of synthetic wastewater was used as bacterial food supply the sludge production was also considerably lower than in conventional treatment [217].
Cox and Deshusses [218] developed a strategy to control biomass growth in
biotrickling filters for waste air treatment by engineering predation of bacteria
by protozoa. It was shown that clogging of bench-scale biotrickling filters could
be slowed down with the use of protozoa. Interestingly, it was found that the reactor with protozoa had a shorter start-up time, possibly because of bacterial
growth factors secreted by the protozoa.
For the biodegradation of whey, the ciliate Tetrahymena had been chosen by
Bonnet at al. [219] as a micro-organism capable of degrading and modifying
the whey biologically in order to diminish its pollutant effect (whey is the
aqueous phase that separates from the curds during cheese making or casein
production). Disposal of crude whey completely arrested operation of lagoon
pilots serving as example of receptor media, whereas the effects of biodegraded
whey were only temporary, and normal operation was recovered within a few
days. The authors stress that this method could be a valuable tool for small
dairy farms, being unable to use complex industrial treatment technologies to
forestall pollution by waste whey.
Clearly, optimal conditions for protozoan activity need to be further evaluated and pilot scale experiments have to be performed to prove the influence of
biomass predators in real treatment systems. Nonetheless these findings are
auspicious, suggesting that specific use of ciliates can be made to improve biodegradation processes.
Protozoa in Wastewater Treatment: Function and Importance
245
4.2
Ciliates as Biosensors
As a constitutive group within the microbial food web, ciliates not only play an
important ecological role in the self-purification and matter cycling of natural
aquatic ecosystems, but also in the artificial system of sewage treatment plants.
Their feeding on bacteria improve the treatment, resulting in higher transparency, i.e. lower organic loads in the output water of the treated wastes (see
Sects. 1 and 2). This status of ciliates as an important functional group, improving the process in municipal sewage treatment, and furthermore that‚ “values
from ciliate growth inhibition tests are relevant for the risk assessment for
sewage treatment plants” has been recently acknowledged by a Technical
Recommendation of the EEC [220].
There is a broad consensus in ecotoxicology that taxonomic similarity (i.e.,
close relationship, in terms of phylogeny) generally implies similar toxicological responses, e.g., [221, 222]. This is reflected in aquatic toxicology by selecting
certain fish, crustacean, and algae species to represent trophic and taxonomic
levels as a whole. A transferability of toxicological data for ciliates is also indicated. Although there exists an extraordinary amount of evolutionary distance
between different genera and even between species of the same genus [223,
224], comparisons between the ciliates Colpidium, Colpoda, Paramecium,
Tetrahymena, Uronema, and Vorticella reveal an almost comparable toxicological susceptibility [210]. Despite the lack of standardized ciliate test protocols,
only 2 substances out of 13 exert a toxic effect differing by a factor of more than
100, whereas for the rest of the chemicals the deviations lie within about one order of magnitude (Fig. 17).
The early use of ciliates in toxicity testing was reviewed by Persoone and Dive
[225]. Among the ciliates, the organism of choice in aquatic toxicity testing has
become the common freshwater hymenostome Tetrahymena [195, 226, 227].
Many features have contributed to making Tetrahymena – particularly the species
T. pyriformis and T. thermophila – favorite models in cell biology and facilitated
their modern day use as aquatic toxicity test organisms. It is worth mentioning,
not only that these unicellular organisms can be grown under axenic, i.e., bacteria-free conditions, but also that they combine important advantages from two
groups of organisms. Indeed, they belong to the higher cells, the eukaryotes, but
they can be cultured both easily and economically like the prokaryotic bacteria.
An innovative tool with the potential of a wide application has recently been
offered by the introduction of a commercialized microtoxicity test kit with
Tetrahymena (Protoxkit F, Creasel Ltd., Belgium). The test is specially designed
for the use of environmental samples, thereby providing a helpful means to assess risks of sewage contaminants and their possible detrimental effects on the
performance of waste water treatment plants. Following the concept of readyto-use microbiotests, with the test kit a ciliate multi-generation (growth) assay
can be conducted by non-experts without sophisticated sample preparation and
expensive equipment.
Growth impairment tests with Tetrahymena have generally reached the highest degree of acceptance and standardization [195, 227, 228]: Based on an inter-
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Fig. 17. Comparison between toxic effects on ciliates from different genera (data from
IUCLID, effect of methanol on T. pyriformis: own measurement). The arrows indicate cases
where the ciliate data deviate by a factor of more than two orders of magnitude, from [210]
national pilot ring test, a growth test with the ciliate Tetrahymena is recommended by the German Federal Environmental Agency for ecotoxicological risk
assessment [229]. A final ring test to establish an internationally recognized Test
Guideline has been initiated – an important step to include a traditionally untested, but ecologically important group of organisms in comprehensive ecotoxicity test batteries.
5
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