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CHAPTER 3

Protozoa in Wastewater Treatment:


Function and Importance
Wilfried Pauli 1, Kurt Jax 2, Sandra Berger 1
1 Institut fr Biochemie und kotoxikologie, Freie Universitt Berlin, Ehrenbergstr. 2628,
D-14195 Berlin, Germany, E-mail: wpauli@zedat.fu-berlin.de
2 Zentrum fr Ethik in den Wissenschaften, Universitt Tbingen, Keplerstrasse 17,
D-72074 Tbingen, Germany

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.

Keywords. Protozoa, Ciliates, Ecology, Sewage treatment, Environmental biotechnology

1 Ecological Role of Aquatic Protozoa with Special Regard


to Ciliates Within the Microbial Food Web . . . . . . . . . . . . . 205
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
1.2 Traditional Food Webs and Microbial Food Webs . . . . . . . . . . 205
1.3 The Role of Protozoa in Aquatic Food Webs . . . . . . . . . . . . . 208
1.4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

2 Protozoa in Wastewater Treatment . . . . . . . . . . . . . . . . . . 212


2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.1.1 Wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.1.2 Biological Treatment Processes . . . . . . . . . . . . . . . . . . . . 214
2.1.3 Bacterial Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.1.4 Activated Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.2 Protozoa in Biological Wastewater Treatment Plants . . . . . . . . 217
2.2.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
2.2.2 Species Composition . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2.2.3 Plant Specific Basic Communities . . . . . . . . . . . . . . . . . . . 220
2.2.4 Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.2.5 Ecological Framework . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.2.5.1 Sludge Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

The Handbook of Environmental Chemistry Vol. 2 Part K


Biodegradation and Persistence
(ed. by B. Beek)
Springer-Verlag Berlin Heidelberg 2001
204 W. Pauli et al.

2.2.5.2 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223


2.2.5.3 pH-Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.2.5.4 O2-Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2.3 Significance of Protozoa for Wastewater Treatment . . . . . . . . . 225
2.3.1 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.3.2 Reduction and Elimination of Suspended Particles and Bacteria . 227
2.3.2.1 Clearing Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
2.3.2.2 Experimental Findings . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.3.2.3 Field-Observations . . . . . . . . . . . . . . . . . . . . . . . . . . 231
2.3.3 Elimination of Dissolved Substances . . . . . . . . . . . . . . . . . 232
2.3.4 Flocculation and Composition of the Bacterial Community . . . . 232
2.3.5 Reduction of the Total Biomass . . . . . . . . . . . . . . . . . . . . 235
2.3.6 Influence of Protozoa on Bacterial Metabolism . . . . . . . . . . . 237
2.3.7 Filamentous Bacteria and Protozoa . . . . . . . . . . . . . . . . . . 239

3 Impairments of Protozoa: Consequences for Water Purification . 241

4 Environmental Biotechnological Aspects . . . . . . . . . . . . . . 243


4.1 Biodegradation Potentials of Ciliates . . . . . . . . . . . . . . . . . 243
4.2 Ciliates as Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . 245

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

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 differ-
ent order (primary consumers, secondary consumers and so on) and decom-
posers [3]. The latter live on the dead organic matter and mineralize the organ-
ic compounds to inorganic nutrients, e.g., phosphorus, nitrogen, etc. These ca-
tegories were also the basis on which Raymond Lindeman [4] built his famous
trophic dynamic concept of ecology which was the first implementation of
Arthur Tansleys 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 Lindemans
parlance were commonly attributed to phytoplankton (producers), zooplank-
ton (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 recog-
nized and used for applied purposes rather early. Protozoa were used as bioin-
206 W. Pauli et al.

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.

Table 1. The size classes of planktonic organisms

Picoplankton Nanoplankton Microplankton Macroplankton

0.22 mm 220 mm 20200 mm > 200 mm


Bacteria Algae Algae Ciliates
Cyanobacteria Flagellates Rhizopods Rotatoria
Algae Rhizopods Ciliates Crustaceans
Flagellates Ciliates Rotatoria Fish larvae
Ciliates Nauplii
208 W. Pauli et al.

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

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
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, re-
spectively). 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 waste-
water 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

213
which the wastewater flows
214 W. Pauli et al.

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 Settleable: 33% (w/v) or


total ca. 450 mg/l) 150 mg/l, 33% (BOD)
Non-settleable: 67% (w/v) Dissolved: 83% (w/v)
or 300 mg/l, 67% (BOD) 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 oxy-
gen demand (COD, BOD) of the organic substances. Furthermore, a differentia-
tion is made between suspended and dissolved wastewater components.
Approximately two-thirds of the total load (organic and inorganic) of muni-
cipal sewage is in solution. With regard to the organic load almost half is in so-
lution, the rest consists of colloidal material (25%) or is bound to particles
which sediment (75%). Similarly, about half of the oxygen demand of biochem-
ically 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 or-
ganic 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 l1 (ca. 450 mg l1
organic content). Activated sludge plants aim for effluent values < 20 mg
BOD5 l1, i.e., a reduction in the organic content of more than 90% [41]. For in-
dustrial 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 ef-
fects.

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 pro-
cesses, 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 discov-


ered 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 or-
ganisms 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 ef-
ficiency [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 (sub-
merged aerator contactors). In this way, a nutrient concentration exceeding the
degradation capacity of local surface waters, resulting in disruption or even de-
struction of natural biological systems, can be avoided: Direct discharge of sub-
stances would result in anaerobic or aerobic burdening of the sediment of sur-
face waters; high oxygen consuming, organic content (BOD5) in the effluent can
overtax the oxygen household of the water, through its rapid conversion by he-
terotrophic 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 sub-
stances 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 na-
tural 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, start-
ing from nutrients through to decomposers and primary producers and both
primary and secondary consumers, has been replaced by a more complex eco-
logical 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 bio-
coenosis 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 waste-
water 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
216 W. Pauli et al.

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

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 pro-
cesses, forming the basis for the biomass layer, precede the attachment of a bio-
film. Dissolved organic molecules (polysaccharides, proteins, humic acids) ac-
cumulate spontaneously on the surface of very different materials forming a
conditioning film, on which bacteria colonization follows. The cells are im-
mobilized 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 bio-
logical investigations than slime-growth areas of fixed-bed reactors on the
other.
Activated sludge processes operate with typical sludge concentrations be-
tween 23 g l1 [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 2025% of the sludge [41].
In a conventional activated sludge tank flocculate suspended material con-
tains about 6 109 bacteria ml1, i.e., 13 1012 bacteria g1 dry weight [48].
They represent about 90% of the total biomass of the activated sludge. The pro-
portion of living or metabolically active bacteria found in the flocs varies con-
siderably, depending on the method of analysis. Estimates based on glucose,
stearate and acetate uptake rates imply active proportions of 813%, 1428%,
and 510% of the total biomass, respectively [48]. More recently, direct mea-
surements by fluorescence-microscopy indicate a proportion of 3540% (de-
Protozoa in Wastewater Treatment: Function and Importance 217

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 re-


veal protozoa as typical components of the biocoenosis (Table 3). Thus, for ex-
ample, 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 com-
munities 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 there-
fore to certain areas of the wastewater treatment plant; only a few specialists
among the protozoa take part in anaerobic processes. Thus protozoan commu-
nities can be typically encountered in activated sludge tanks as well as in the se-
dimentation tanks, whereas no protozoa are found in sludge digestion or in the
supernatant of the sedimentation tank (effluent), with the exception of malfunc-
tions [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 [5255]

Type of plant No. of plants Occurrence of Typical Protozoa


investigated typical protozoan protozoan absent
(country) communities communities
absent

Activated sludge 56 (Great Britain) Within 53 plants 2 plants a 1 plant


39 (Italy) Within 38 plants b 1 plant b ?
10 (South Africa) Within all 10 plants
Trickling filter 52 (Great Britain) Within all 52 plants
Rotating biological 49 (Italy) Within 47 plants b 2 plants b ?
contactor
10 (USA) Within all 10 plants
a No ciliates, but flagellates present.
b Only ciliates investigated, no comments on other protozoan groups such as flagellates and
amoebae.
218 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 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]

Plant 1 Plant 2 Plant 3

Organic load a 0.230.38 0.210.35 0.50.8


O2-conc. (mg O2/l) 3.65.2 1.83.0 1.01.3
Densities and biomass ind./ml mg/l ind./ml mg/l ind./ml mg/l
Ciliates 3000 1843 8600 5099 4500 2693
7400 17 000 16000
Flagellates (< 20 mm) 43 000 2.25.2 89000 4.651 38000 2083
600 000 980 000 1 600 000
Naked Amoebae (<50 mm) 4000 0.215.3 800 0.04 77000 4.15.4
100 000 130 000 6.9 101 000
a kg BOD5/(kg MLVSS) day.
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 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

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 fi-
gure) and ca. 80 days (RBC, below figure), respectively. Bars lower than 100% indicate the ad-
ditional 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 succes-
sion 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 ob-
served, dependent on the filter depth [68].
Figure 5a, b shows results from studies on the colonization behavior of cili-
ates 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 1015 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 45% of the slime bio-
mass in the colonization phase, as opposed to 1219% under steady-state con-
ditions. 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. Investiga-
tions 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 consider-


ed as a measure for its significance within the ecological structure of the bio-
logical 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 percola-
tion filter plants in Northern Italy. Of note is that the British and Italian activat-
ed 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 de-
termined 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 ml1, ciliate densities are 1001000 times higher
Protozoa in Wastewater Treatment: Function and Importance 221

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

In activated sludge plants a high proportion of the eukaryotic biomass is com-


prised 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 99100% 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 mu-
nicipal 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
222 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 Life form Nutrition a


(%) (%) Ecological type
GB I GB I

Activated sludge (GB and I)


Aspidisca costata a 35 85 69 90 Crawling Bacterivorous
Vorticella convallaria a 19 77 58 84 Sessile Bacterivorous
Trachelophyllum pusillum a 15 30 64 58 Free swimming Carnivorous
Opercularia coarctata a 12 23 54 25 Sessile Bacterivorous
Carchesium polypinum a 11 26 25 28 Sessile Bacterivorous
Vorticella alba (GB) 11 38 Sessile Bacterivorous
Vorticella microstoma (GB) 10 10 75 10 Sessile Bacterivorous
Euplotes moebiusi (GB) 5 5 35 7 Crawling Bacterivorous
Vorticella fromenteli (GB) 4 31 Sessile Bacterivorous
Euplotes affinis (I) 59 11 69 Crawling Bacterivorous
Zoothamnium pygmaeum (I) 33 33 Sessile Bacterivorous
Trochilia minuta (I) 2 23 12 25 Crawling Filamentous b
Trickling filter (GB)
Opercularia micodiscum 44 81 Sessile Bacterivorous
Carchesium polypinum 15 62 Sessile Bacterivorous
Vorticella convallaria 10 83 Sessile Bacterivorous
Chilodonella uncinata 4 90 Crawling Filamentous b
Opercularia phryganeae 4 90 Sessile Bacterivorous
Opercularia coarctata 2 56 Sessile Bacterivorous
Vorticella striata 2 52 Sessile Bacterivorous
Aspidisca costata 56 Crawling Bacterivorous
Cinetochilum margaritaceum 54 Crawling Bacterivorous
Rotating biological contactor (I)
Euplotes moebiusi 53 79 Crawling Bacterivorous
Paramecium caudatum 46 79 Free swimming Bacterivorous
Trachelophyllum pusillum 41 59 Free swimming Carnivorous
Vorticella convallaria 53 57 Sessile Bacterivorous
Opercularia microdiscum 41 45 Sessile Bacterivorous
Opercularia coarctata 33 37 Sessile Bacterivorous
Paramecium trichium 27 43 Free swimming Bacterivorous
Cinetochilum margaritaceum 23 37 Crawling Bacterivorous
Chilodonella cucullulus 18 41 Crawling Filamentous b
a Dominant both in British and Italian plants.
b Filamentous: ciliates with a specialized oral apparatus, enabling the ingestion of rod-shap-
ed, filamentous bacteria.
not present.
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-chem-
ical 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 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

Conventional processes of biological wastewater treatment utilize the meta-


bolism 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 an-
aerobic degradation processes. The number of facultative anaerobic protozoa is
slightly higher, but almost all species seem to be able to survive low oxygen con-
centrations 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 organ-
isms 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 ses-
sile 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 ci-
liates 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 be-
come 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 [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.

Fig. 8. Mechanisms of filter-feeding (ambiguously often referred to as grazing) used by pro-


tozoa. Water currents are created by flagella or the coordinated activity of cilia, that bring sus-
pended food to the mouth region of the cell

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

Table 6. Effects of ciliated protozoa on the effluent quality of bench-scale activated-sludge


plants. Results are given in mg l1 unless otherwise noted; after [103]

Effluent analysis Without ciliates With ciliates Mean reduction

BOD 5370 724 75%


COD 198250 134142 38%
Permanganate value (4 h) 83106 6270 30%
BOD after filtration 3035 39 81%
COD after filtration 3150 1425 39%
Organic nitrogen 1421 710 51%
Suspended solids 86118 2634 71%
Optical density at 620 nm 0.951.42 0.230.34 76%
Viable bacteria counts (106 ml1) 160 19 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 unci-


nata, 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 bio-
coenosis, the authors show that a largely selective reduction of protozoa, by the
addition of sodium fluoride (0.2 mol l1) or sodium azide (620 mmol l1) re-
sults in a notably higher content of freely suspended bacteria including strep-
tococci. After application of the eukaryotic cell toxins, the total count of fecal
streptococci increases about threefold and the proportion of suspended bac-
teria, 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 bigua-
nide hydrochloride) with known effects on bacteria and protozoa were studied
and a causal relationship between poor quality of the outflow (increased turbi-
dity and COD values) from batch cultures of activated sludge and the inhibitory
effect (reduction in population density) on the protozoa was observed. A corre-
lation 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: 0.10.4 g BOD per g MLSS and day) show that the selective de-
cline of the ciliate population density, due to running temperatures of 36C and
over (see Sect. 2.2.5.2), corresponds to a more than twofold increase in suspen-
ded 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.
230 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 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.

Finally, as compared to normal activated sludge processes, the clarifying ef-


fect of protozoa in activated sludge processes with submerged fixed-bed filters
a technology which creates additional surfaces for slime growth and primar-
ily sessile ciliates [116118] 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 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

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 [132134]. However, wastewater itself contains a high proportion of
chemically complex particles of differing sizes, and bacteria themselves, domi-
nant with regard to their biomass in wastewater treatment plants, produce ex-
tracellular polymeric substances (polysaccharides), to which they can effec-
tively adsorb [135, 136]. For these reasons, protozoa, by excretion of digested re-
mains 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 pre-
dator-prey relationship between protozoa and bacteria, a collapse of the bacte-
ria 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 obser-
ved in model systems [111, 137141] and in natural ecosystems [20, 142146]
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 waste-
water 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 ap-
paratus for highly efficient filtration, which at the same time exclude particles
of several micrometers in diameter [86]. The ability of bacteria to develop larg-
er forms, to grow collectively, or to merge as micro-colonies protects them
against the predator pressure from the protozoa [148153]. 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 spe-
cies to larger or aggregating phenotypes [45, 149], cannot be decided in the light
of the present literature. Gde [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., phe-
notypes 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 proto-
zoa autochthonous aerobic bacteria and cultures of Salmonella typhimurium
and E. coli introduced into the sewage sludge are predominantly freely suspend-
ed (4368%). In the presence of protozoa, the proportion of freely suspended
234 W. Pauli et al.

bacteria drops significantly to 115%. The majority can now be found in or


adhering to flocs (8599%); 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: 50C, 5 min) se-
wage 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 fil-
ter-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 ni-
ches (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]
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

Doubling time (h) Temperature ( C)

Activated sludge a 3.310 20


Aspidisca costata b 13.6 20
Aspidisca lynceus b 12.4 20
Vorticella microstoma b 5.0 20
Vorticella convallaria b 7.6 20
Carchesium polypinum b 9.3 20
Opercularia spec b 5.0 20
Epistylis plicatilis b 10.2 20
Colpidium campylum b 4.7 20
Tetrahymena pyriformis b 4.5 20
Paramecium caudatum b 12.0 20
a [158].
b [111].
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 dis-
solved 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 circum-
stances 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, in-
creases [128, 161]. This diminished ability to utilize available nutrients for bio-
mass 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 ml1 and over [61, 75, 9497]. The number of bacteria required to maintain
this ciliate population can be estimated based on data from Macek [162]. Under
steady-state conditions (20C) and generation times of 5 days, free-swimming
ciliates such as Colpidium campylum and sessile forms such as Vorticella micro-
stoma at densities of 1.3 104 ml1 and 0.59 104 ml1 consume, over the 5-day
period, 2.5 109 and 2.1 109 bacteria ml1 (450 and 420 mg COD l1), respec-
tively. The bacterial content of sewage arriving at the plant is on average
106 ml1. 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 den-
sity of 104 ml1 would require more than 17 106 bacteria ml1 and hour (23
105 ml1 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 con-
tent 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 0.35 mm [8587; 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 li-
pids, 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 un-
clear 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 ty-
pical 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 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

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 maintain-
ed or even increased: In bench-scale plants, ciliates show no effects on the nitrifi-
cation 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].
238 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 pre-
sence of protozoa, an increased degradation (BOD, COD) of the dissolved, non-
filterable portion of organic materials, attributed almost exclusively to the ac-
tivated 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 degra-
dation processes with regard to the organic pollutants 2,4-dichlorophenol (2,4-
DCP) 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 cul-
tures after 15 days the environmental chemicals 2,4-DCP and 2,4-D were min-
eralized in the presence of protozoa to 70% and 90%, respectively: Whereas in
cultures where the protozoa were inhibited by nystatin and cycloheximide, de-
gradation 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 at-
tempts emphasize the direct influence of the protozoan metabolic activity;
others attach more importance to bacteria feeding and its indirect consequen-
ces 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 me-
tabolizing 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 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

other hand, protozoa release a series of organic substances such as amino-acids


[170] and growth factors, having chemical structures not characterized in de-
tail [22, 109, 171175], 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 advantag-
eous interaction by nitrogen and phosphorus re-mineralization is emphasized
by many authors [22, 145, 177183]. To what extent these additional organic and
inorganic substances introduced into the wastewater cycle play a part in waste-
water 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 ra-
tio 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 car-
bon 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 turn-
over. A possible cause could be the qualitative shifting of the selection condi-
tions for the bacteria and therefore the composition of mixed bacteria popula-
tions 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 feed-
ing-resistant bacterial growth forms can be viewed mainly as a result of pha-
gocytic activity of protozoa: Freely suspended bacteria are succeeded by aggre-
gated, 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 adsorp-
tion and metabolism of organic compounds. At low densities, they contribute to
240 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 pro-
cesses in the sedimentation tank which can, in extreme cases, due to the forma-
tion of light, fluffy, poorly settling flocs, result in the discharge of sludge into na-
tural 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 treat-
ment are adjusted to low carbon concentrations [low F/M ( food:micro-orga-
nism) 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, pa-
per 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 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.

the 50% lethal effect concentrations of Cu<Hg<Cd<Pb<Cr<Zn (1 mg l1


50 mg l1) on various ciliates isolated from activated sludge, whereby the au-
thors report differences in species sensitivities of up to two orders of magni-
tude, dependent on the heavy metal tested. Kakiichi et al. [107, 198200] 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 as-
pera 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 indi-
cator 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, 203209] 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 respi-
ration test; out of a total of 32 substances, just 6 cases had a (toxic) effect po-
tential of less than 100 mg l1. The weak correlation of r2 = 0.17 confirms the dis-
crepancy between the two tests (Fig. 15). Similar observations of a low correla-
tion 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, 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
References
1. Berninger U-G, Finlay BJ, Kuuppo-Leinikki P (1991) Limnol Oceanogr 36:139
2. Jax K (1992) Limnologica 22:299
3. Thienemann A (1926) Verh Dtsch Zool Ges 31:29
4. Lindeman RL (1942) Ecology 23:399
5. Tansley AG (1935 Ecology 16:284
6. Bick H (1989) kologie. Gustav Fischer, Stuttgart
7. Kolkwitz R, Marsson M (1908) Ber Dtsch Bot Ges 26a:509
8. Kolkwitz R, Marsson M (1909) Int Rev Gesamten Hydrobiol 2:126
9. Bick H (1964) Die Sukzession der Organismen bei der Selbstreinigung von organisch
verunreinigtem Wasser unter verschiedenen Milieubedingungen. Habil-Schr, Dssel-
dorf
10. Bick H (1973) Am Zool 13:149
11. Pomeroy LR (1974) BioScience 24:499
Protozoa in Wastewater Treatment: Function and Importance 247

12. Porter KG, Paerl H, Hodson R, Pace M, Priscu J, Riemann B, Scavia D, Stockner J (1988)
Microbial interactions in lake food webs. In: Carpenter SR (ed) Complex interactions in
Lake Communities. Springer, Berlin Heidelberg New York, p 209
13. Graham JM (1991) J Protozool 38:66
14. Sieburth JM, Smetacek V, Lenz J (1978) Limnol Oceanogr 23:1256
15. Pomeroy LR, Wiebe WL (1988) Hydrobiologia 159:7
16. Stockner JG, Antja NJ (1986) Can J Fish Aquat Sci 43:2472
17. Stockner JG, Porter KG (1988) Microbial food webs in freshwater planktonic ecosystems.
In: Carpenter SR (ed) Complex interactions in lake communities. Springer, Berlin
Heidelberg New York, p 69
18. Sherr EB, Sherr BF (1991) Trends Ecol Evol 6:50
19. Porter KG, Pace ML, Battey JF (1979) Nature 277:563
20. Sanders RW, Porter KG, Bennet SJ, DeBiase AE (1989) Limnol Oceanogr 34:673
21. Sanders RW (1991) J Protozool 38:76
22. Bloem J, Br-Gilissen M-JB (1989) Limnol Oceanogr 34:297
23. Pace ML, Orcutt JDJ (1981) Limnol Oceanogr 26:822
24. Beaver JR, Crisman TL (1982) Limnol Oceanogr 27:246
25. Beaver JR, Crisman TL (1989) Microb Ecol 17:111
26. Ducklow HW, Purdie DA, Williams PJL, Davies JM (1986) Science 232:865
27. Gifford DJ (1991) J Protozool 38:81
28. Pace ML, McManus GB, Findlay SEG (1990) Limnol Oceanogr 35:795
29. Carrick HJ, Fahnenstiel GL, Stoermer EF, Wetzel RG (1991) Limnol Oceanogr 36:1335
30. Hamilton FT, Taylor WD (1987) Can J Fish Aquat Sci 44:1038
31. Currie DJ, Kalff J (1984) Limnol Oceanogr 29:298
32. Buechler DG, Dillon RD (1974) J Protozool 21:339
33. Caron DA, Goldman JC, Dennett MR (1988) Hydrobiologia 159:27
34. Schnborn W (1982) Limnologica 14:329
35. Baldock BM, Sleigh MA (1988) Arch Hydrobiol 111:409
36. Harmsworth GC, Sleigh MA, Baker JH (1992) J Protozool 39:58
37. Lock MA, Wallace RR, Costerton JW, Ventullo RM, Charlton SE (1984) Oikos 42:102
38. Christensen ST, Wheatley DN, Rasmussen MI, Rasmussen L (1995) Cell Death and
Differentiation 2:301
39. Neill WE (1994) Spatial and temporal scaling and the organization of limnetic commu-
nities. In: Giller PS, Hildrew AG, Raffaelli DG (eds) Aquatic ecology: scale, pattern and
process. Blackwell Scientific, Oxford, p 189
40. Ppel F (1980) Lehrbuch fr Abwassertechnik und Gewsserschutz. Deutscher Fach-
schriften, Wiesbaden
41. Abwassertechnologie (1988) Deutsche Gesellschaft fr Technische Zusammenarbeit
(ed) Springer, Berlin Heidelberg New York
42. Bhnke B (1980) Wissenschaft Umwelt 1:27
43. Schlegel S (1995) Korrespondez Abwasser 8:1343
44. Meyer-Reil L-A (1994) Mar Ecol Prog Ser 112:303
45. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Annu
Rev Microbiol 49:711
46. Winkler M (1981) Biological treatment of waste-water. Ellis Horwood, Chichester
47. Hartmann L (1989) Biologische Abwasserreinigung Springer, Berlin Heidelberg New York
48. Pike EB (1975) Aerobic bacteria. In: Curds C R, Hawkes HA (eds) Ecological aspects of
used-water treatment. Academic Press, London, p 1
49. Griebe T, Schaule G, Secker J, Flemming H-J (1996) Bestimmung der stoffwechselaktiven
Bakterien im Belebtschlamm. In: Lemmer H, Griebe T, Flemming H-C (eds) kologie
der Abwasserorganismen. Springer, Berlin Heidelberg New York, p 155
50. Wagner M, Amann R (1996) Die Anwendung von in situ-Hybridisierungssonden zur
Aufklrung von Struktur und Dynamik der mikrobiellen Bioznosen in der
Abwasserreinigung. In: Lemmer H, Griebe T, Flemming H-C (eds) kologie der
Abwasserorganismen. Springer, Berlin Heidelberg New York, p 93
248 W. Pauli et al.

51. Wagner M, Amus B, Hartmann A, Hutzler P, Amann R (1994) J Microsc 176:181


52. Bux F, Kasan HC (1994) Water S A (Pretoria) 20:61
53. Curds RC, Cockburn A (1970) Water Res 4:225
54. Chung JC, Strom PF (1991) Res J Water Pollut Control Fed 63:35
55. Madoni P, Ghetti PF (1981) Hydrobiologia 83:207
56. Varma MM, Finley HE, Bennett GH (1975) WPFC J 47:85
57. Sydenham DHJ (1971) Hydrobiologia 38:553
58. Hughes DE, Stafford DA (1976) Critical Reviews in Env Control 6:233
59. Curds CR (1982) A Rev Microbiol 36:27
60. Madoni P (1982) Acta Hydrobiol 24:223
61. Aescht E, Foissner W (1992) Arch Hydrobiol 90:207
62. Salvado H (1994) Water Res 28:1315
63. Kinner NE, Curds CR (1987) Water Res 21:481
64. Madoni P (1994) Water Sci Technol 29:63
65. Madoni P (1994) Bioresource Technology 48:245
66. Curds RC, Cockburn A (1970) Water Res 4:237
67. Mudrack K, Kunst S (1994) Biologie der Abwasserreinigung. G Fischer, Stuttgart
68. Curds CR (1992) Protozoa and the water industry. Cambridge University Press,
Cambridge
69. Klee O (1968) Ger Mikrokosmos 57:231
70. Klimowicz H (1970) Acta Hydrobiol 12:357
71. Salvado H, Gracia MP (1993) Water Res 27:891
72. Salvado H, Gracia MP (1994) Verh Internat Verein Limnol 25:1950
73. Eikelboom DH (1988) Extra toepassingsmogelijkheden voor protozoa en metazoa bij de
zuivering van afvalwater. TNO, Delft, Nr R88/286
74. Lee EGH, Mueller JC, Walden CC (1975) Tappi 58:100
75. Curds CR (1975) Protozoa. In: Curds CR, Hawkes HA (eds) Ecological aspects of used-
water treatment. Academic Press, London, p 203
76. Toman M, Rejic M (1988) Z f Wasser- und Abwasserforschung 21:189
77. Hill DL (1972) The biochemistry and physiology of Tetrahymena. Academic Press,
London
78. Hoffmann EK, Rasmussen L (1972) Biochim Biophys Acta 266:206
79. Orias E, Rasmussen L (1979) J Cell Sci 36:343
80. Rasmussen L, Zdanowski MK (1980) Experentia 36:1044
81. Seaman GR (1955) Metabolism of free-living ciliates. In: Hutner SH, Lwoff A (eds)
Biochemistry and physiology of protozoa, vol 2. Academic Press, London, p 91
82. Cirillo VP (1962) J Bacteriol 84:754
83. Andersen AP, Hellung-Larsen P (1989) J Cell Biochem 41:125
84. Nilsson JR (1979) Phagotrophy in Tetrahymena. In: Lewandowski M, Hutner S (eds)
Biochemistry and physiology of protozoa, 2nd edn, vol 2. Academic Press, New York, p
339
85. Sleigh M (1989) Protozoa and other protists. Edward Arnold, London
86. Fenchel T (1980) Microb Ecol 6:1
87. Fenchel T (1980) Microb Ecol 6:13
88. Holz GG (1973) The nutrition of Tetrahymena: essential nutrients, feeding, and diges-
tion. In: Elliott AM (ed) Biology of Tetrahymena. Dowden Hutchinson, Stroudsburg, p 89
89. Rasmussen L, Modeweg-Hansen L (1973) J Cell Sci 12:275
90. Fenchel T (1986) Progr Protistol 1:65
91. Hatzis C, Sweeney PJ, Srienc F, Fredrickson AG (1993) Biotechnol Bioeng 42:284
92. Seaman GR (1961) J Protozool 8:204
93. Curds RC, Cockburn A (1968) J Gen Microbiol 54:343
94. Baines S, Hawkes HA, Hewitt C H, Jenkins SH (1953) Sewage Indust Wastes 25:1024
95. Ministry of Technology (1968) Not Wat Pollut 43:1
96. Bark AW (1972) Annls Stn Limnol Besse 67:241
97. Augustin H, Foissner W, Bauer R (1989) Acta Hydrochim Hydrobiol 17:375
Protozoa in Wastewater Treatment: Function and Importance 249

98. Curds CR (1971) Wat Res 5:1049


99. Ardern E, Lockett WT (1928) Manchester Rivers Dept Ann Rep 1:41
100. Pillay SC, Subrahmanyan V (1942) Nature 150:525
101. Pillay SC, Wadhwani TK, Gurbaxani MI, Subrahmanyan V (1944) Nature 154:179
102. McKinney RE, Gram A (1956) Sewage Ind Wastes 28:1219
103. Curds RC, Cockburn A, Vandyke JM (1968) Wat Pollut Control 67:312
104. Sridhar MKC, Pillai SC (1974) Environ Pollut 6:195
105. Macek M (1991) Single-species ciliate cultures controlling bacterial flocs distribution.
In: Madoni P (ed) Proc Int Symp Biol Approach to Sew Treatment Process. Perugia
1990, p 109
106. Farrah SR, Scheuerman PR, Eubanks RD, Bitton G (1985) Water Sci Technol 17:165
107. Kakiichi N, Kamata S, Ito O, Komine K, Otsuka H, Uchida K (1991) Anim Sci Technol
(Jpn) 62:32
108. Curds CR, Fey GJ (1969) Wat Res 3:853
109. Mallory LM, Yuk CS, Liang LN, Alexander M (1983) Appl Environ Microbiol 46:1073
110. Pitman AR (1975) Water Pollut Control 74:688
111. Sudo R, Aiba S (1984) Adv Biochem Eng 29:117
112. Esteban G, Tellez C, Bautista LM (1990) Environ Technol 12:381
113. Fernandez-Leborans G, Moro P (1991) Bioresour Technol 38:7
114. Salvado H, Gracia MP, Amigo JM (1995) Water Res 29:1041
115. Sasahara T, Ogawa T (1983) Monatsschrift fr Brauwissenschaft 11:443
116. Middeldorf JM (1989) Korrespondenz Abwasser 10:1165
117. Schlegel S (1986) Wasser Abwasser 127:421
118. Hu HY, Fujie G, Urono K (1993) Wat Sci Technol 28:179
119. Schlegel S (1988) Korrespondenz Abwasser 2:120
120. Reilly M (1964) J Protozool 12:109
121. Weekers PHH, Vogels G D (1994) Journal of Microbiological Methods 19:13
122. Glaser D (1988) Microb Ecol 15:189
123. Hrudey SE (1982) J Water Pollut Control Fed 54:1207
124. Witthauer DP (1980) European J Appl Microbiol Biotechnol 9:151
125. Clarholm M (1984) Microbes as predators or prey. In: Klug MJ, Reddy CA (eds) Current
perspectives on microbial ecology. American Society for Microbiology, Washington, DC,
p 321
126. Macek M, Hartmann P, Skopov I (1993) Int Revue ges Hydrobiol 78:557
127. Macek M, Hartmann P (1991) Stud Environ Sci 42 (Environ Biotechnol):113
128. Ratsak CH, Kooi BW, Verseveld HW van (1994) Water Sci Technol 29:119
129. Taylor WD, Berger J (1980) Microb Ecol 6:27
130. Curds CR (1963) PhD Thesis, London University
131. Curds CR (1963) J Gen Microbiol 33:357
132. Fletcher M, Loeb GI (1979) Appl Environ Microbiol 37:67
133. Costerton JW (1992) Int Biodeter Biodegrad 30:123
134. Lappin-Scott HM, Costerton JW (1995) Biofouling 1:323
135. Decho AW (1990) Microbial exopolymer secretions in ocean environments: their role(s)
in food webs and marine processes. In: Barnes M (ed) Oceanogr Mar Biol Annu Rev,
vol 28. Aberdeen University Press, p 73
136. Stehr G, Zrner S, Bttcher B, Koops HP (1995) Microb Ecol 30:115
137. Gause GF (1935) J Exp Biol 12:44
138. Curds CR (1970) Proc Symp on Methods of Study of Soil Ecology. UNESCO, Paris,
France
139. Watson PJ, Ohtaguchi K, Fredrickson AG (1981) J gen Microbiol 122:323
140. Swift ST, Najita IY, Ohtaguchi K, Fredrickson AG (1982) Biotechnol Bioeng 24:1953
141. Huber HC, Huber W, Ritter U (1990) Zbl Hyg 189:511
142. Hapte M, Alexander M (1975) Microbiol 29:159
143. Hapte M, Alexander M (1977) Microbiol 113:181
144. Ibanez F, Rassoulzadegan F (1977) Ann Inst Oceanogr 53:17
250 W. Pauli et al.

145. Clarholm M (1981) Microb Ecol 7:343


146. Fenchel T (1982) Mar Ecol Prog Ser 9:25
147. Fairbrother TH, Renshaw A (1922) J Soc chem Ind Lond 41:134
148. Gde H (1979) Microb Ecol 5:225
149. Shikano S, Luckinbill LS, Kurihara Y (1990) Microb Ecol 20:75
150. Sime-Ngando T, Bourdier G, Amblard C, Pinel-Alloul B (1991) Microb Ecol 21:211
151. Jrgens K, Stolpe G (1995) Freshwater Biol 33:27
152. Jrgens K, Pernthaler J, Schalla S, Amann R (1999) Appl Environ Microbiol 65:1241
153. Sommaragu R, Psenner R (1995) Appl Environ Microbiol 61:3457
154. Gurijala KR, Alexander M (1990) Appl Environ Microbiol 56:1631
155. Kjelleberg S (1984) Effects of interfaces on survival mechanisms of copiotrophic bac-
teria in low-nutrient habitats. In: Klug MJ, Reddy CA (eds) Current perspectives in
microbial ecology. ASM, Washington, p 151
156. Jenkins SH (1942) Nature 150:607
157. Imhoff K (1993) Taschenbuch der Stadtentwsserung, 28 Aufl. Oldenburg, Mnchen
Wien
158. Horan NJ (1990) Biological wastewater treatment systems, theory and operation. Wiley,
Chichester
159. Schnborn (1992) Arch Protistenkd 141:181
160. Calow P (1977) Biol Rev 52:385
161. Pirt SJ (1965) Proceedings of the Royal Society B 163:224
162. Macek M (1989) Int Rev Ges Hydrobiol 74:643
163. Kiy T, Tiedke A (1992) Appl Microbiol Biotechnol 37:576
164. Pauli W, Khnel S, Berger S (1995) Neue biotechnologische Verfahren zur Gewinnung
von Wertstoffen: Grundlagenuntersuchungen fr die berfhrung von Verfahren zur
Erzeugung von Wertstoffen mittels Ciliaten in den klein- bzw. halbtechnischen Mastab.
Abschlubericht zum Forschungsvorhaben des Bundesministers fr Forschung und
Technologie (Frderkennzeichen 0317383B)
165. Lang H (1981) Wasserwirtschaft 71:166
166. Eberhard H (1984) Wasserwirtschaft 74:47
167. Wiggins BA, Alexander M (1988) Can J Microbiol 34:661
168. Elliott AM (1973) Biology of Tetrahymena. Dowden, Hutchinson and Ross, Stroudsburg,
Pennsylvania
169. Chudoba J (1985) Wat Res 19, 197200
170. Andersson A, Lee C, Azam F, Hagstrom A (1985) Mar Ecol Prog Ser 23:99
171. Hervey RJ, Greaves JE (1941) Soil Sci 51:85
172. Nicoljuk VF (1969) Acta Protozool 7:99
173. Taylor GT, Iturriaga R, Sullivan CW (1985) Mar Ecol Prog Ser 23:129
174. Fenchel T (1988) Ann Rev Ecol Syst 19:19
175. Henkinet R, Couteaux M-M, Billes G, Bottner P, Palka L (1990) Soil Biol Biochem 22:555
176. Sherr BF, Sherr EB, Hopkinson CS (1988) Hydrobiologia 159:19
177. Barsdate RJ, Prentki RT, Fenchel T (1974) Oikos 25:239
178. Woods LE, Cole CV, Elliot ET, Anderson RV, Coleman DC (1982) Nitrogen transforma-
tions in soil as affected by bacterial-microfaunal interactions. Soil Biol Biochem 14:93
179. Bloem J, Starink M, Br-Gilissen MJB, Cappenberg TE (1988) Appl Environ Microbiol
54:3112
180. Coleman DC (1985) Through a ped darkly: an ecological assessment of soil-root-micro-
bial-faunal interactions. In: Fitter AH, Atkinson D, Read DJ, Usher MB (eds) Ecological
interactions in soil. Blackwell, Oxford, p 1
181. Coleman DC, Crossley DAJ, Beare MH, Hendrix PF (1988) Agric Ecosyst Environ 24:117
182. Fenchel T, Harrison P (1976) The significance of bacterial grazing and mineral cycling for
the decomposition of particulate detritus. In: Anderson JM, MacFadyen A (eds) The role
of terrestrial and aquatic organisms in decomposition processes. Blackwell, Oxford, p 285
183. Rutherford PM, Juma NG (1992) Can J Soil Sci 72:217
184. Ratsak CH, Maarsen KA, Kooijman SALM (1996) Wat Res 30:1
Protozoa in Wastewater Treatment: Function and Importance 251

185. Lemmer H (1996) Ursachen und Bekmpfung von Blhschlamm. In: Lemmer H Griebe
T, Flemming H-C (Hrsg) kologie der Abwasserorganismen. Springer, Berlin Heidelberg
New York
186. Suwa Y, Imamura Y, Suzuki T, Tashiro T, Urushigawa Y (1994) Wat Res 28:1523
187. Alldredge AL, Cole JJ, Caron DA (1986) Limnol Oceanogr 31:68
188. Jeffrey WH, Paul JH (1986) Appl Environ Microbiol 51:1177
189. Karner M, Herndl GJ (1992) Mar Biol 113:341
190. Seguchi K, Koga M (1983) Proceedings of the 20th Annual Meeting of Sewage Works
Researches. Tokyo, Japan
191. Hashimoto R (1985) J Jpn Sewage Works Assoc 22:61
192. Nitta T, Sakai Y, Mori T (1987) Appl Microbiol Biotechnol 26:195
193. Inamori Y, Kuniyasu Y, Sudo R, Koga M (1991) Water Sci Technol 23:963
194. Gilron GL, Lynn DH (1997) Ciliated protozoa as test organisms in toxicity assessment.
In: Wells PG, Lee K, Blaise C (eds) Microscale testing in aquatic toxicology. CRC Press,
Boca Raton
195. Sauvant MP, Pepin D, Piccini E (1999) Chemosphere 38:1631
196. Gracia MP, Salvado H, Rius M, Amigo J-M (1994) Acta Protozool 33:219
197. Madoni P, Davoli D, Gorbi G (1994) Bull Environ Contam Toxicol 53:420425
198. Kakiichi N, Kamata S, Komine K, Uchida K (1989) Jpn J Zootech Sci 60:857
199. Kakiichi N, Matsui M, Kamata S, Komine K, Ito O, Hayashi M, Otsuka H, Uchida K (1990)
Jpn J Zootech Sci 61:924
200. Kakiichi N, Yamamoto T, Kamata S, Otsuka H, Uchida K (1993) Anim Sci Technol (Jpn)
64:1013
201. Yoshioka Y, Nagase H, Ose Y, Sato T (1986) Ecotox Environ Saf 12:206
202. OECD (1983) OECD Guideline for Testing of Chemicals Activated Sludge, Respiration
Inhibition Test Draft 1.8.83, No 210
203. Guhl W (1987) Korrespondenz Abwasser 34:1076
204. Poole J E P A (1987) Water Pollut Control 86:116
205. Luna-Pabello V M, Mayen R, Olvera-Viascan V, Saavedra J, Duran De Bazua C (1990)
Biological Wastes 32:81
206. Al-Shahwani SM, Horan NJ (1991) Water Res 25:633
207. Esteban G, Tellez C (1992) Water Air Soil Pollut 61:185
208. Ratsak CH, Kooi BW, Kooijman B (1995) J Euk Microbiol 42:268
209. Martin-Cereceda M, Serrano S, Guinea A (1996) FEMS Microbiology Ecology 21:267
210. Pauli W, Berger S (1999) A new Toxkit microbiotest with the protozoan ciliate Tetra-
hymena. In: Persoone G, Janssen C, de Coen W (eds) New microbiotests for routine toxi-
city screening and biomonitoring. Kluwer Academic/Plenum Publishers, New York, p 169
211. Jaworska JS, Schultz TW (1994) Ecotoxicol Environ Safety 29:200
212. Curds CR, Cockburn R (1971) J Gen Microbiol 66:95
213. Jost JL Drake JF, Frederickson AG Tsuchia HM (1973) J Bacteriol 113:834
214. Ashby RE (1976) J Exp Mar Biol Ecol 24:227
215. Drake JF, Tsuchia HM (1977) Appl Environ Microbiol 34:18
216. Lee NM, Welander T (1996) Biotechnol Lett 18:429
217. Lee NM, Welander T (1996) Wat Res 30(8):17811790
218. Cox HHJ, Deshusses MA (1997) Annual Meeting and Exhibition of the Air and Waste
Management Association. Toronto, Canada
219. Bonnet JL, Bogaerts P, Bohatier J (1999) Chemosphere 38:2979
220. ECB (1988) Effects assessment for micro-organisms in sewage treatment plants: consi-
deration of protozoa toxicity data. Document European Chemicals Bureau 4/TR1/98,
Technical Recommendation, TGD chap 3, sect 4
221. Suter GW (1982) Extrapolation of ecotoxicity data: choosing tests to suit the assessment
CONF-8210487 Environmental Protection Agency, USA
222. Volmer J, Krdel W, Klein W (1988) Chemosphere 17:1493
223. Schlegel M, Eisler K (1996) Evolution of ciliates. In: Hausmann K, Bradbury PC (eds)
Ciliates, cells as organisms. Gustav Fischer, Stuttgart
252 W. Pauli et al.

224. Brunk CF, Kahn RW, Sadler LA (1990) J Mol Evol 30:290
225. Persoone G, Dive D (1978) Ecotoxicol Environ Safety 2:105
226. Schultz TW (1996) Tetrahymena in aquatic toxicology: QSARs and ecological hazard as-
sessment. In: Pauli W, Berger S (eds) Proceedings of the International Workshop on a
Protozoan Test Protocol with Tetrahymena in Aquatic Toxicity Testing. Umweltbundes-
amt-Texte 34/96, Berlin, Germany, p 31
227. Gilron GL, Lynn DH (1997) Ciliated protozoa as test organisms in toxicity assessment.
In: Wells PG, Lee K, Blaise C (eds) Microscale testing in aquatic toxicology. CRC Press,
Boca Raton
228. Pauli W, Berger S (1996) Proceedings of the International Workshop on a Protozoan Test
Protocol with Tetrahymena in Aquatic Toxicity testing. Umweltbundesamt-Texte 34/96,
Berlin, Germany
229. Heger W, Jung S, Martin S, Rnnefahrt I, Schiecke U, Schmitz S, Teichmann H, Peter H
(1998) Chemikaliengesetz Heft 11, kotoxikologische Testverfahren mit aquatischen
Organismen. Texte 58/98, Umweltbundesamt, Berlin, Germany

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