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Protozoa constitute a major link between the highly productive and nutrient retaining micro-
bial 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 im-
portant grazers of microbes in aquatic environments, balancing bacterio-plankton produc-
tion. 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 spe-
cies 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 infor-
mation regarding adverse effects of environmental chemicals on this part of the biocoenosis
essential for the effective operation of biological waste-water treatment processes.
5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
List of Abbreviations
BOD(5) biological oxygen demand (index: within 5 days)
COD chemical oxygen demand
dw dry weight
fm 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 m3; concentration
of the suspended solids in an aeration tank including inorganic mat-
ter)
MLVSS Mixed-liquor volatile suspended solids (g m3; corresponds to the or-
ganic, i.e., combustible content of the sludge, which amounts to ca.
70% of the sludge solids: 0.7 MLSSMLVSS; 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 205
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 pre-
sent in high numbers of species and individuals both in the oceans and in fresh-
water habitats. Some taxa live attached to solid substrates or within the sedi-
ment, some as part of the plankton. An overview of the data about the abun-
dance 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 flagel-
lates (< 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 en-
countered [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 dy-
namics, namely the fluxes of energy and material. It is the objective of this pa-
per 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
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 stu-
dies on this topic were made by Bick and co-workers (e.g., [9, 10]) who investi-
gated the succession of micro-organisms, in particular ciliated protozoa, in the
course of the self-purification of water enriched with sewage and other organ-
ic 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 micro-
organisms are supposed to play within aquatic systems. These insights were in-
itiated by the appearance of some new actors on the stage of the ecological thea-
ter 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 devel-
oped in connection with marine systems they were soon transferred to fresh-
water 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.
Protozoa in Wastewater Treatment: Function and Importance 207
By the early 1970s it was recognized that an important part of the pelagic or-
ganisms 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 abun-
dance of bacteria in the open water had been underestimated by orders of mag-
nitude. Only 0.11% of the actual abundance had been counted [12]. Fur-
thermore, most investigations of marine and freshwater plankton used plank-
ton 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 consist-
ing 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 ma-
terial 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 ear-
lier, 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 condi-
tions and 1670% in fresh waters [16]. For oligotrophic lakes 5070% are do-
cumented, while the autotrophic picoplankton amounts to 1045% of the total
phytoplankton biomass (standing stock, measured as chlorophyll) [17]. Data for
marine habitats give estimates of 2080% [18]. Similarly, the abundance of he-
terotrophic picoplankton, i.e., heterotrophic bacteria, is much higher than pre-
viously 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 knowl-
edge.
Fig. 2. The food web of the lake plankton. The classical food chain (open circles) is supple-
mented by the elements of the microbial loop (filled ovals and square). DOM: dissolved or-
ganic 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 he-
terotrophic 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 tra-
ditional 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) with-
in 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 dur-
ing 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 sys-
tems seems obvious. They are not only in the same way as bacteria decom-
posers associated with the decay of organic material, but they are a link between
Protozoa in Wastewater Treatment: Function and Importance 209
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 mate-
rial into edible portions and thus make it available to crustaceans, rotatoria, and
other metazoans. There is empirical evidence that planktonic protozoa graze ef-
fectively 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 with-
in 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, he-
terotroph, mixotroph, etc.) which does not correspond with the classic taxonom-
ic or trophic level boundaries [21]. Furthermore the body sizes of the differ-
ent taxa are important features for their position within the food webs.
In many cases bacteria are grazed upon mainly by small heterotrophic fla-
gellates, 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 re-
lationships between these groups.
HNAN are sometimes grazed upon directly by metazoa, while in other bo-
dies of water ciliates constitute the main predators [17, 23]. Heterotrophic fla-
gellates, possessing high turnover rates, inhabit a central position in the trans-
fer of organic carbon in most microbial food webs.
But what about the ecological roles of ciliates? In some cases, especially in pro-
ductive 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 (2030 mm) were
largely excluded from lakes having <5 106 5 108 bacteria ml1 a concen-
tration normally found only in more productive systems. Large ciliates
(> 50 mm), being mainly phytophagous and grazing on nanoplanktic algae, do-
minate the ciliate assemblages in oligotrophic lakes, with low bacterial abun-
dance. Mixotrophic ciliates with endosymbiotic algae can even contribute sub-
stantially to pelagic autotrophic biomass in some lakes (15% of annual total [25]).
The overall number of planktonic ciliates in lakes is correlated with the tro-
phic state of the water bodies.While under oligotrophic conditions abundancies
210 W. Pauli et al.
of 310 cells ml1 were recorded, 90215 cells ml1 were recorded in hypereu-
trophic 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 mate-
rial. 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 depen-
dent 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 he-
terotrophic flagellates to larger ciliates and then to crustaceans or other meta-
zoans. In other cases crustaceans may directly feed on nanoplankton, while ci-
liates 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 com-
partment 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 211
As mentioned above, the microbial loop is not only important for the trans-
fer of energy in the form of organic carbon, but also for the cycling and reten-
tion 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 gra-
zers. Algae and bacteria compete for P, with bacteria being more efficient in the
uptake of P. Bacterial grazing by protozoa was demonstrated to enhance phos-
phorus turnover and mineralization [30]. As grazed bacteria populations grow
faster their excretion of P also becomes stronger. Furthermore, protozoan gra-
zers increase the amount of organic P by excretion, which seems to be of spe-
cial importance for phytoplankton [31]. Although this compound is also excret-
ed 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 as-
semblage, 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 out-
compete 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 al-
lows an increase in algal biomass [33]. The degree to which this process is of im-
portance depends on the carbon available for the bacteria. As Caron et al. [33]
concluded: the role of bacterivorous protozoa as mineralizers of a growth-
limiting 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 re-
lated 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 [3436], our understanding of the complex web of interre-
lations 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
212 W. Pauli et al.
assemblages. Thus we are only just beginning to delve deeper into the compli-
cated patterns and dynamics of those biofilms. There is now important evi-
dence 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 in-
volved 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 abio-
tic 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 especi-
ally 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
213
which the wastewater flows
214 W. Pauli et al.
2.1.2
Biological Treatment Processes
with bacteria they form a closely related microbial system which forms the ba-
sis of the so-called natural self-purification process.
2.1.3
Bacterial Biofilms
2.1.4
Activated Sludge
2.2
Protozoa in Biological Wastewater Treatment Plants
2.2.1
Occurrence
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 [5255]
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 pro-
portion with regard to biomass and number of species, both in activated sludge
[53, 5762] and in fixed-bed processes (percolation filters: [53, 59]; rotating disc
contactors: [6365]), compare Table 4.
It should be noted, however, that the composition of the protozoan biocoe-
nosis, as well as that of the total biomass involved in the purification process,
is mainly dependent on the composition of the wastewater, together with phy-
sical conditions and factors arising from the process technology used. In the
case of malfunctions, or in the initial stage of a plant, very different composi-
tions 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 612 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 vi-
sible on the disc surfaces within one day of startup followed within a few days
by flagellates and small amoebae. Free-swimming bacterivorous ciliates appear-
ed within 810 days. Subsequently, sessile peritrichous forms accompanied by
carnivorous ciliates, rotatoria, and large amoebae make up the stable commu-
nity. 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 12 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]
ming ciliates predominate, more and more crawling and sessile forms appear,
which remain dominant throughout the stabilization phase and can be regard-
ed as typical representatives of mature sludge [62, 65, 6870]; 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
2.2.3
Plant Specific Basic Communities
Fig. 6. Examples of free swimming (holotrichous), crawling (hypotrichous), and sessile (pe-
ritrichous) ciliates in waste water treatment plants
here, than in the plankton of oligotrophic (10 ml1) and eutrophic (100 ml1)
waters [24]. Table 5 summarizes the dominant ciliate species in the basis com-
munity 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 in-
crease 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
2.2.5
Ecological Framework
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
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 ac-
tivated sludge plants (e.g., [47, 67]). Ciliate densities of 600030,000 ml1 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 mu-
nicipal 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
0.11.4 [g BOD (g MLVSS day)1]. Only under very heavy loads [1.82.4 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 domi-
nant 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 = 0.10.3 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 = 0.30.6, peritrichous spe-
cies dominate and by high organic loads of fm = 0.60.9 equal portions of peri-
trichs and holotrichs are present (Fig. 7).
2.2.5.2
Temperature
Temperatures in municipal plants are generally slightly above the outside tem-
perature in winter and slightly below in summer. Performance is optimal be-
tween 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 al-
kaline effluents are introduced, mainly from industrial processes, pH-values
generally fluctuate between 6.5 and 8 [41, 67, 75]. Therefore not only the tem-
224 W. Pauli et al.
Fig. 7. Composition and species number of ciliates in activated sludge plants operated at dif-
ferent 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 holo-
trichous ciliates represent sessile, crawling, and free swimming ciliates, respectively. In con-
ventional 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
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 ses-
sile ciliates Vorticella convallaria and Opercularia coarctata, typically found in
wastewater treatment plants [76].
2.3
Significance of Protozoa for 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 [7779], di-peptides [80], acetate, glucose [81,
82], and even for such complex nutrient solutions as proteose-peptone-yeast ex-
tract (PPY) medium [83]. Another method of nutrient uptake is pinocytosis
[84, 85]. It describes the active transport of dissolved substances in sub-micro-
scopic, 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 mat-
ter 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.
with the aid of a ciliary sieve, which retains in the case of bacterivorous spe-
cies particles sized between 0.3 mm and 5 mm [8587]. The particles, thus con-
centrated, are subsequently ingested. Apart from food, abiotic and even indige-
stible matter of the size of bacteria are efficiently ingested [8689]. 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 5080 nl is cleared of particles per hour and cell [87, 91],
a more than 1000 times lower water volume of 36 pl h1 and cell is actually in-
gested by the food vacuoles [92]. The efficiency of this form of nutrition is un-
derlined by investigations comparing the growth kinetics of Tetrahymena pyri-
formis with particulate and dissolved substances as nutrient source, respec-
tively [93]. While under monoxenic conditions with particulate bacterial
substrate the half maximum growth rate is already attained with a bacteria con-
tent of 12 mg l1 Klebsiella aerogenes (5.5 mg carbon l1), 200 times that con-
centration of organic matter is required in case of dissolved nutrients (2.4 g l1
proteose-peptone-yeast medium = 1.3 g carbon l1).
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 h1 at temperatures between 9C and 17C [20]. Higher filtration rates are
observed for larger ciliates. Sanders et al. [20] quote a yearly fluctuation range
of 12156 nl h1 for the filtration performance of planktonic ciliates. In labora-
tory experiments with the ciliates Halteria grandinella (diameter: 25 mm) and
Strombidium sp. (size: 15 21 mm) filtration rates of 8090 nl h1 at 9C and
120140 nl h1 at 17C were determined. In the case of Vorticella microstoma
(average cell dimensions: 60 30 mm), a ciliate frequently present in wastewa-
ter treatment plants, filtration rates as high as 156 nl h1 at bacteria densities of
106 ml1 are reported. Tetrahymena (cell dimensions: 40 20 mm), a species
present but not dominant in wastewater treatment plants, has a filtration per-
formance of 80 nl h1 [91]. Fenchel [87] observed filtration rates of 50 nl h1 and
cell at 2022C for Tetrahymena pyriformis and 2001000 nl h1 for larger
(100200 mm) representatives of crawling and free-swimming ciliates such
as Euplotes, Paramecium, or Blepharisma. Assuming average filtration rates of
100 nl h1 and cell and ciliate densities of 10,000 ml1 and above [61, 9497], 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 2040 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 divi-
sion 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 = 0.050.6 g BOD per g MLSS and day), growth rates can vary from 450%
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 com-
pensate for potential protozoan feeding.
Highly loaded wastewater contains ca. 106 bacteria ml1. The majority are
medically harmless but others are pathogenic and bear health risks. Con-
ventional wastewater purification involves an initial pre-clarification step of
2030 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
24 h from the treated wastewater in a separate, specifically designed sedimen-
228 W. Pauli et al.
tation tank. The supernatant from the separation stage is the treated waste-
water, 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 con-
centration 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 sedi-
mentation 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 ac-
tual 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 treat-
ment plants over a long period. Through the subsequent re-introduction of ty-
pical sludge ciliates they observed, under various starting conditions, positive
effects on a series of parameters describing the success of the purification pro-
cess (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 ac-
tivated sludge. The addition of sessile, crawling, and free-swimming ciliates
bacterial density
Fig. 9. Influence of ciliates on the bacteria content in the effluent of a bench-scale activated
sludge plant, after [103]
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 ml1
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 ten-
fold to 6.3 104 ml1. 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 in-
fections 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 l1) or
sodium azide (20 mmol l1), cultures of Salmonella typhimurium and E. coli, ad-
ded in densities of 105 ml1 almost treble within 24 h (S. typhimurium and so-
dium 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 stabiliza-
tion, the authors show that even low densities of protozoa (660 ml1) lead to a
substantial elimination of bacteria. Figure 10 shows results with Streptococcus
faecalis.
Fig. 10. Effect of sodium azide (6 mmol l1, 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 sus-
pended matter in the effluent of wastewater treatment plants (domestic and
municipal wastewater [56, 110114] 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 signifi-
cant correlation coefficient between total ciliate population density and biolog-
ical oxygen demand of r = 0.868. In the presence of protozoa the effluent BOD
ranges from 4 mg l1 to 18 mg l1, rising to values of up to 67 mg l1 in their ab-
sence. 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 l1 were found with ciliate densities of ca. 104 ml1; these in-
crease to 40 mg l1 when ciliate densities drop to 102 ml1 (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.
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 treat-
ment plants is negligible: For these substances the essential activity comes from
the bacteria population. They dominate the biomass and possess a higher me-
tabolic efficiency as a result of their high surface to volume ratio [41, 4648, 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 imme-
diate rise in the bacterial biomass was observed, whereas ciliates were scarcely
able to convert the available peptone into their own biomass and could only re-
produce 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 con-
tributing 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, 124128].
This is attributed, on the one hand, to polymer, particle-aggregating excre-
tion products (polysaccharides) from protozoa [59, 125], which are possibly re-
leased into the media to facilitate a more effective uptake of particles [24, 129].
Protozoa in Wastewater Treatment: Function and Importance 233
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]
Protozoa in Wastewater Treatment: Function and Importance 235
2.3.5
Reduction of the Total Biomass
In order not to exceed a sludge concentration favorable for the purification per-
formance of the wastewater treatment plant, an amount equal to the daily pro-
duction must continuously be drawn off. This excess sludge is subsequently
concentrated, digested, and drained and must finally be disposed of as a poten-
tially pathogenic and frequently toxic waste product. Excess sludge is therefore
an economic factor, even within the wastewater treatment plant itself. A reduc-
tion in sludge production corresponds to savings in personnel, energy, and run-
ning costs.
Since the function of the sedimentation tank is merely to separate the bio-
mass from the purified water, the effluent concentration must already be attain-
ed 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 810 days [157]. This means that the sludge
biomass doubles after 4 days, at the earliest. Generation times in this range im-
ply 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 515 h; see Table 7.
In principal, a lengthening of the food chain results in a reduction of the orig-
inally 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-mineraliza-
tion and less biomass production.
Table 7. Doubling times of activated sludge and ciliates isolated from activated sludge plants
concentrate and ingest particulate matter the size of bacteria from the sur-
rounding 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 be-
tween 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 al-
most exclusively bacterial biomass was supplied with sterile synthetic waste-
water 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 23 g (dw)
l1 [46], which corresponds to ca. 6 109 bacteria ml1 [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 activat-
ed sludge consume 1.52.9 109 bacteria ml1. In other words, even at shorter
retention times in a plant aimed primarily at the elimination of carbon com-
pounds, a considerable proportion (2548%) of the bacteria can be phago-
cytosed by ciliates: This corresponds to a 1019% reduction of the accumula-
ted 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 pro-
tozoa. 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 sub-
merged fixed-beds are primarily colonized by protozoa whereby ciliates domi-
nate [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
Fig. 13. Effects of protozoa on the mineralization of 0.1 mg l1 2,4-dichlorophenol and 2,4-
dichlorophenoxyacetic acid (2,4-D) in sewage. Cycloheximide (250 mg l1) and nystatin
(30 mg l1) were used to suppress protozoa; from [167]
Protozoa in Wastewater Treatment: Function and Importance 239
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 adsorp-
tion and metabolism of organic compounds. At low densities, they contribute to
240 W. Pauli et al.
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 pre-
dacious protozoan Trochilioides recta; from [194]
Protozoa in Wastewater Treatment: Function and Importance 241
tent, stimulates the development of bulking sludge. Various chemical (e.g., lim-
ing, chlorination, addition of H2O2 , iron salts, and nitrogen and phosphorus
compounds) and physical (e.g., increased oxygen supply) methods are imple-
mented to combat bulking. Sometimes even operational conditions of plants
are altered (e.g., increasing the return-flow rate, by-passing the pre-clarifica-
tion, 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 operatio-
nal plants [190193]; see Fig. 14. Effective cell densities for Trochilioides sp. are
quoted as 1000 ml1 [190] and for Trithigmostoma cucullus and Trochilioides
recta as 2000 ml1 [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 per-
formance of plants. Excess influx of toxic wastes with detrimental effects on ci-
liates 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., EC50-
values ranking from some mg l1 to some g l1 (reviewed by [194, 195]). Sub-
stances 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 metal-
finishing 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 sub-
stances 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 con-
centrations of 1 mg Cu2+ l1 on species diversity and population density espe-
cially of the ciliates of natural sludge samples. Madoni et al. [197] determined
242 W. Pauli et al.
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]
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, 212215]. 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 pro-
tozoa 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 pre-
dation (see Sect. 2.3.4), dispersed bacteria can be readily taken up and meta-
bolized 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 1243% in biomass yield in comparison with a system without ci-
liate 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 3050% of the yields typically obtained in
conventional aerobic processes [216]. If authentic instead of synthetic waste-
water was used as bacterial food supply the sludge production was also con-
siderably 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 re-
actor 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 evaluat-
ed 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 bio-
degradation 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 trans-
parency, 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, improv-
ing 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 toxicologi-
cal 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 indi-
cated. 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 toxicologi-
cal 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 or-
der 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., bac-
teria-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 as-
sess risks of sewage contaminants and their possible detrimental effects on the
performance of waste water treatment plants. Following the concept of ready-
to-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 high-
est degree of acceptance and standardization [195, 227, 228]: Based on an inter-
246 W. Pauli et al.
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 recom-
mended 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 un-
tested, but ecologically important group of organisms in comprehensive eco-
toxicity test batteries.
5
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