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Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A.
Méndez-Vilas (Ed.)
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Progresses on the knowledge about the ecological function and structure
of the protists community in activated sludge wastewater treatment plants
L. Arregui1, B. Pérez-Uz1, H. Salvadó2 and S. Serrano1
1
2
Department of Microbiology III, Complutense University of Madrid, C/ Jose Antonio Novais 2, 28040 Madrid, Spain
Department of Animal Biology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
Occurrence and density of microbial populations, and particularly of the protists community, are directly related to the
operational parameters performance of wastewater treatment plants (WWTP) such as sludge retention time or effluent
quality. Microscopic monitoring of floc structure and other biological factors as filamentous bacteria and protist
populations, have become a frequent practice to evaluate the state of the purification processes; however, the application of
biotic indexes is not as accurate as it would be expected. The microorganism species cited in different WWTP studies are
very restrained if these communities are compared to those described in natural waters. ”In vivo” observations using
conventional or phase contrast optical microscopy are the most frequent method employed to identify protist species
although precise identification is difficult to achieve in this way. The inaccurate assignment of species to other similar
morphotypes following previous observations or reports in depuration processes is unfortunately too frequent in the plant
laboratories. Besides, many specimens go undetected due difficulties such as small size, colorless cytoplasm, the presence
of an agglutinate test or a lorica included within the flocs, etc. Therefore it would be feasible an increase on protists
diversity if an accurate identification was performed. The use of general silver staining techniques or, more recently, of the
fluorescent taxoid FLUTAX has facilitated solving some of the problems listed. In addition, most studies in the literature
dealing with protist communities have been carried out on conventional activated sludge systems although more efficient
activated sludge plants new designs are continuously developing in response to the legislation requirements concerning the
removal of carbonaceous, nitrogen and phosphate compounds. These advanced nutrient removal systems combine aerobic,
anoxic and anaerobic phases with distinct strategies of sludge recirculation. As it will be discussed, these treatments
restraints are responsible to shape singular protist communities present in the biological reactors, becoming flagellates and
amoebae usual components of the floc populations sharing this niche with typical crawling ciliate species.
Keywords WWTP; protist communities; ciliates; flagellates; amoebae; silver staining; FLUTAX
1. Introduction
Aerobic biological treatment systems use the metabolic activity of chemoorganoheterotrophic microorganism
communities for the degradation of organic compounds, through aerobic respiration processes. Wastewater treatment
plant designs facilitate the development of a diverse and stable microbial community which in an aerated environment
assures the efficient removal of organic matter allowing as well the clarification of the treated water.
The activated sludge system facilitates the aggregation of microorganisms that are embedded within or in the surface
of a complex heterogeneous structure named floc. Flocs are freely suspended in the mixed liquor. Treated water reaches
then the clarification tank where flocs are separated as sludge from treated water by settling, allowing the clearing up of
the effluent. Besides, settled sludge is used in these biological processes as a reinoculation of the biological reactor [1].
Flocs are, therefore, the functional and operative units of these systems, having different sizes and texture depending on
the environmental conditions. Flocs having inadequate dimensions, with a loose structure, disaggregated or with a
filamentous aspect do not settle properly, producing inconveniences in the effluent clarification and deteriorating the
depuration effectiveness [2].
Biological communities within the reactors of activated sludge plants are mainly composed by microorganisms.
Major components of this ecosystem are bacteria reaching 90-95% of the biomass. Bacteria can be found free or
forming part of the floc (isolated, grouped or in a filamentous form). These populations are responsible of the
biodegradation of organic material and the removal of toxic contaminants; they are also the primary colonizers of the
reactor during the first steps of the process and, as previously mentioned, are involved in the floc formation [3]. Protists
and other microorganisms represent 5-10% of the biological total biomass [4]. Protist community is involved in the
predation of bacterial populations and in the flocculation process allowing as well obtaining an efficient effluent
clarification [5, 6, 7].
2. Floc structure
The analysis of the floc structure is becoming a frequent practice within the parameter control protocols performed by
WWTP operators in order to prevent certain plant performance problems, or to solve alterations already present. Floc
macrostructure is determined by the presence of filamentous bacteria that provide the primary matrix needed for the
settlement of floc-forming bacteria. However, an overgrowth of filamentous bacteria might cause serious problems of
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Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
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bulking giving place to inefficient settlement of flocs and therefore ineffective clarification stages. The floc
microstructure is constituted by different bacterial and protists populations and some microinvertebrates that remain
attached in the aggregate by biological secreted exopolimeric substances [2] (Fig.1).
The “Grupo de Bioindicación de Sevilla” (GBS) developed in 2008 a Sludge Index (SI), based in certain flocular
characteristics as a control parameter [8]. This index has been successfully applied by different wastewater plant
laboratories in Spain. This index assigns a score to several macroscopical characteristics of the sludge such as turbidity,
presence of suspended flocs, odour and sedimentability - detected with the V30 assay- and other microscopical features
as floc size, morphology, compactness, firmness and number of interflocular filamentous bacteria. Other aspects
associated to the floc macrostructure are also evaluated including the presence of microbial suspended cells, protists
diversity and the presence/absence of organic fibers and/or inorganic particles. Therefore a microscopical analysis along
with the sedimentability assay information allows to obtain a numerical value (between 0-100), providing an evaluation
tool to assess the activated sludge quality. This tool can be used to modify the working conditions in the biological
treatment processes in order to improve their performance.
b)
a)
c)
Fig. 1 Floc structure. a) Schematic draw of the floc macrostructure, b) compact floc larger than 500 µm and c) floc with open
structure and interflocular filamentous bacteria.
3. Protist populations in activated sludge reactors
Protist communities present in the aeration tanks characterize the biological conditions of the plant. In fact, a stable
protist community is common in steady state conditions with a set up of operational and design parameters in any
efficient WWTP. Environmental changes in these conditions are immediately revealed by a modification of the
community structure [7, 9, 10, 11].
Nowadays, conventional activated sludge systems are being replaced by other advanced designs for the elimination
of nutrients to avoid deterioration of the ecological quality of waters by eutrophication, as the stricter legislation and
control from the EU Water Framework Directive (2000/60/EC) rules. It has been demonstrated that these WWTPs
designed for nutrient elimination show protist communities with different abundances, composition and activity
compared to those found in conventional plants [12, 13, 14]. Therefore, previous studies about the established
biocenosis in conventional activated sludge plants cannot be directly extrapolated to these new types of WWTP.
3.1
Protist populations in conventional systems (Fig. 2a)
Madoni (1993) proposed that certain community characteristics should be used as criteria to define an efficient
activated sludge plant [15], among these:
- High numbers of microfauna cells (> 106 organisms/l);
- Microfauna composed mainly by crawling and attached ciliates, with almost no flagellates;
- Highly diversified species and ciliate groups and none dominating numerically over the others by a factor
greater than 10.
Ciliates in this context were traditionally considered the most important populations of the protist communities in the
aeration tanks of WWTP. Curds [6] and Madoni [9, 16] classified ciliates in four ecological categories depending on
their nutrition strategies, type of locomotion and physical location within the floc:
- free-swimming ciliates; those no associated to the floc, frequently bearing cilia uniformly distributed over the
cell, as Paramecium sp or Uronema nigricans.
- crawling ciliates; those moving on the floc surface, with flattened cells and specialized ventral ciliature mainly
hypotrichs such as Euplotes and Aspidisca species.
- attached ciliates; those firmly fixed to the floc through a stalk (peritrichs as Vorticella convallaria or Epistylis
species), a lorica (as Thuricola sp or Vaginicola sp), etc.
- carnivorous ciliates; those free-swimming or sessile ciliates (suctorids) predators of flagellates or other ciliates.
Curds et al. (2008) [17] have also recently illustrated 175 ciliate species considered as “true” sewage ciliates although
more than 200 have been reported previously in activated sludge processes. However, only a fraction of these can be
commonly observed in individual plants.
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Amoebae (naked or testate amoeba) and flagellates have been described in these systems as protist associated to the
colonization stages or to inefficient depuration processes.
3.2
Protist populations in advanced nutrient removal systems (Fig. 2b)
WWTPs with enhanced nutrient biological removal processes are being used nowadays for the depuration of urban,
agricultural and industrial wastewaters. Removal of nitrogen and phosphorus compounds is possible by combined
systems with aerobic, anoxic and anaerobic stages (by compartmentalizing one reactor or different reactors with distinct
strategies of sludge recirculation) [18]. These treatments restraints are responsible to shape the microbiota in charge of
the purifying process and therefore the protist community present in these biological reactors is also different as it has
already been observed by different authors [12, 13, 14].
Interestingly, ciliate population in the oxic stage of plants with a good nitrification performance shows a very low
abundance compared to other protist populations. In these systems, amoebae and flagellates are found to represent
stable populations within the biological reactor.
The stable protist community could be described then as follows [13]:
a. Stable populations associated to flocs, include:
a.1. Protists physically associated to flocs through fixation structures and feeding on other organisms found in the
mixed liquor, among these the following groups are typical:
- Heterotrophic nanoflagellates (HNF), mainly the bodonid group. These have characteristically two flagella using
the recurrent larger one to fix themselves to the floc surface. These microorganisms are mainly bacterivorous,
feeding on suspended bacteria found in the mixed liquor.
- Bacterivorous sessile ciliates such as peritrichs, attached through stalks with some representative species as
Opercularia articulata, Epystilis chrysemidis or species of the Vorticella aquadulcis complex.
- Loricated ciliates attached to the flocs through the lorica, usually bacterivorous species, such as the peritrichs
Thuricola and Vaginicola or the prostomatid Metacystis.
- Carnivorous suctorid ciliates fixed by stalks and feeding on other protists.
a.2. Protist associated to flocs through their biological activities, mainly due to type of movement and/or feeding
strategies:
- Naked and testate amoebae, grazing on floc bacteria, with higher abundances especially in small amoeba
species (< 50 µm).
- Crawling ciliates: community mainly composed by few species of litostomates (being Acineria uncinata the
most representative) and phylopharingids (mostly Trochilia minuta) usually also grazing on bacteria.
Hypotrichs as Aspidisca sp. are also present.
- Carnivorous ciliates such as several small size species of the genus Holophrya, adhered to extracellular
polymeric substances components of the floc, feeding on other protist found in the mixed liquor.
b. Transitional populations, found in the mixed liquor.
- Free-swimming, large colourless flagellates, mostly euglenids (ie. Peranema, Entosiphon, Petalomonas,
Notosolenus).
- Naked and testate amoebae that can feed over free or flocculating bacteria.
a)
Fig. 2
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b)
Diversity of ciliates in activated sludge systems. a) Conventional WWTP and b) Advanced nutrient removal WWTP.
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3.3
Biotic indexes
Several researchers have proposed different biotic indexes to evaluate quickly and easily the state of the depuration
process in WWTP. The most popular indexes have been the Shannon index and the Sludge Biotic Index; they both
provide information about the structure of the ecosystem. The Shannon index measures the diversity observed within a
particular ecosystem; the units of measurement are bits [19]. It has been a useful tool to supply an idea about the
performance and the organic loading rate for conventional processes [20]. This index has also been used by our group in
ENBR (Enhance Nutrient Biological Removal) systems where values below 1 indicate inefficient processes and
between 2 and 3 indicate the most efficient processes.
The sludge biotic index (SBI) of Madoni [9] also evaluates the biological functioning in activated sludge plants.
This index is based on the microscopic identification and the estimation of protists abundance and, in particular, of
small and large flagellates, testate amoebae and groups and species of ciliates. This index is actually employed by
almost all plant controllers.
Both indexes involve identification and counting of protists species. “In vivo” observations using conventional or
phase contrast optical microscopy are the most frequently used methods to identify species. These procedures are
however unsuitable, in general, to achieve precise species identification. Therefore is possible that protist species
diversity might increase if further methodologies for an accurate identification are performed.
4. Identification of species diversity in activated sludge biological reactors
The correct characterization of species appearing with high frequency in each plant is decisive in order to assess the
diversity within this artificial ecosystem. Once a species is well identified it could be easily recognized in the daily
controls without any additional methods (Fig. 3).
b)
a)
h)
i)
j)
c) d)
e)
k)
l)
m)
f)
g)
n)
o)
Fig. 3 Protist diversity in activated sludge. Flagellates: a) Peranema sp and b) small flagellates. Amoebae: c) testate amoeba
Arcella sp and d) small naked amoeba. Ciliates: e) Holophrya sp, f) Paramecium Aurelia, g) Trochilia minuta, h) Vorticella
aquadulcis complex, i) Pseudovorticella elongata, j) Epistylis plicatilis, k) Opercularia coarctata, l) Tokophhrya quatripartita, m)
Acineria uncinata, n) Euplotes affinis and o) Aspidisca cicada.
4.1
Flagellates identification
The most representative groups found in activated sludge are the heterotrophic nanoflagellates (HNF) from the
kinetoplastid bodonid group and the larger colourless euglenids of genera such as Peranema (Fig. 3a), Entosiphon or
Petalomonas. Identification has been traditionally performed through morphological analysis “in vivo”. FLUTAX
procedure has rendered as well very good results. The main benefits of this procedure are its efficiency, simplicity of
performance and fast results. This method was initially used to identify ciliates species present in water samples since
direct visualization of the infraciliature of ciliated protozoa is easily achieved [21]; however, flagellate specimens can
also be detected within wastewater samples since this fluorescent taxoid binds to the microtubular systems of these
organisms as well [22]. It is especially useful for the detection of specimens difficult to spot due to their small size that
are included within the floc (Fig. 3b). Number of flagella, size and cell location of these microtubular structures as well
as other basic morphological characteristics used for flagellate identification, are revealed with this methodology.
Ideally, small flagellates should be identified with electron microscopy (both scanning electron microscopy or
transmission electron microscopy whole mounts) although the methodologies for preparation and observation are very
complex and not easy to apply in a routine protocol in a WWTP site.
4.2
Amoebae identification
Species identification of ameboid protists from wastewater samples (and/or other biotopes) is still a problem since some
of the most frequently found naked amoeba families show limited morphological features under light-microscopy to
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allow precise species identification (Fig. 3c,d). Due to this difficulty only few researches have considered deeply these
groups, however, the key to the identification of gymnamoeba by Page (1988) [23] is a helpful tool for microscopic
examination. The development of reliable molecular tools for species identification should be a priority in this group.
The molecular diversity of amoebae until now has been studied only in a few genera (Acanthamoeba, Vanella, Nebela)
based almost exclusively on SSU rRNA gene sequences. More recently, the mitochondrial gene encoding for
cytochrome c oxydase subunit I (COI) has been proposed as a possible good candidate for DNA barcoding of amoeba
[24]. The aforementioned molecular techniques have nowadays no applicability in the daily control of the WWTP.
4.3
Ciliates identification
As ciliates dominate the protist community of activated sludge conventional systems, much work has been carried out
in order to quantify and identify them. Detailed descriptions of the most important ciliates present in these plants can be
found in several excellent guides [25, 26, 27, 28, 29, 30, 31]; however, species identification is still difficult and the
assignment of species to those typical morphotypes already described in previous works about depuration processes is
probably frequent in plant laboratories.
4.3.1
Common problems to identify ciliate species
A number of ciliate species can be identified in vivo by employing phase contrast or bright field microscopy (ie.
Suctoria, Fig. 3l). Peritrichs as Vorticella (Fig. 3h) or Epistylis (Fig. 3k) have some morphological characters such as
the macronuclear position, the shape and size of the zooid and stalk that allow the identification of some species. Others
characters such as the infraciliature in both, vegetative cells and telotroch larvae, must be revealed by staining
procedures and should be employed if more specific information is needed. For example, to identify some peritrichs is
necessary to visualize the cortical argyrome, i.e. to avoid the confusion between the species Vorticella convallaria and
Pseudovorticella elongata (Figs. 3i and 4b).
Other ciliates, especially prostomatids, phylopharingids, some oligohymenophores and litostomates are not easy to
differentiate just with in vivo observations. Several examples are mentioned below:
- Ciliates with an apical-subapical cytostome, a clearly visible cytopharynx and a terminal vacuole could be
considered as prostomatid ciliates but it is impossible with just these data to discern the species. The application of
staining procedures allow several morphological structures to be recognized: the circumoral ciliature composed by
dikinetids at the apical extreme of the somatic ciliature, cortical folds around the cytostome, a strong cytopharynx, close
to 50 longitudinal somatic kineties and a caudal cilium. These data allow differentiating clearly three different ciliate
species: (1) Plagiocampa rouxi, (2) Holophrya teres and (3) Holophrya discolor (Figs. 3e, 4a).
- Acineria uncinata (Fig. 3m) and Litonotus sp are litostomates usually very common in biological reactors. They
have laterally flattened bodies and are lanceolated in shape; infraciliature data are also necessary for correct species
identification.
- Phyllopharyngids as Trochilia minuta could be identified “in vivo” because of the presence of a posterior cell
protrusion or spine, although the staining of the infraciliature is advisable (Fig. 3e). However, two other genera –
Chilodonella and Pseudochilodonopsis- can be easily confused because their in vivo morphology is very similar. In this
case, the staining procedure can reveal the continuous or fragmented preoral kinety that allows the undisputable
identification (Fig. 4c).
- The Oligohymenophores are generally identified to species level with the identification of their infraciliary pattern
(both oral and somatic ciliature: oral structures, number of somatic kineties, development of kinetodesmal fiber) and the
size and shape of the cells.
- Spirotrichs, especially sticotrichs such as Euplotes affinis (Fig. 3n) or Aspidisca cicada and hypotrichs can be
identified “in vivo”. Infraciliary pattern or cortical characteristics might be necessary in some cases though (Fig. 3o).
a)
b)
c)
Fig. 4 Protist identification. a) Holoprya discolor stained with silver carbonate, scale bar: 10 µm; b) Pseudovorticella elongata
stained with Klein, scale bar: 10 µm; and c) Pseudochilodonipsis fluviatilis stained with FLUTAX method, scale bar: 50 µm.
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4.3.2
Methods
- FLUTAX method [21]
The use of FLUTAX can be particularly useful for revealing specimens that are within a lorica and at the same time, are
attached within the flocs. These features usually prevent their observation with other methods, however the most
frequently employed techniques in samples exhibiting diverse ciliate populations involve the use of different silver
salts. These techniques that have been mentioned in the previous section will be briefly described next:
Table 1
FLUTAX procedure
Product
Sample
Saponine (0,5% in PHEM)
Paraformaldehyde (2% in PHEM)
Flutax 1µM
Quantity
500 µl
1 drop
1 drop
10 µl
The sample (500 µl) is set in a depression slide followed by the addition of saponine. After 30 seconds, cells are fixed
for 1 minute with one drop of paraformaldehyde. Several fixed specimens must be then captured with a micropipette
and included within a 10 µl FLUTAX drop placed on a slide. The observations can be carried out immediately after
placing a coverslip in a fluorescence microscope.
- Silver carbonate method [32, 33]
Silver carbonate is a fast and easy method to use (20 min). The main disadvantage of this technique is the use of
pyridine which implies to carry out the staining process under a fume hood to avoid the pyridine vapours. This staining
process evidences different structures: somatic and oral infraciliary pattern, nuclear composition (macro and
micronuclei) and other cortical characteristics.
The procedure is summarized as described below (Table 2) All reagents should be mixed up consecutively in a small
beaker:
Table 2
Silver carbonate method procedure.
Volume
2 ml
2 drops
2ml
25-30 drops
10 drops
2 ml
30 ml
Product
Sample
Formalin (40%)
Tween 80 (5%)
Bactopeptone
Piridine (pure)
Ammoniacal argentic solution
Distilled water
Once the sample has been mixed with the reagents, the beaker is heated at 60º C in a water bath during 10 minutes
until the sample-reagents mix colour changes to dark brown. Then the staining mix is poured in a capsule with 20 ml of
cold distilled water. Cells are then left to settle (5-10 minutes) and the overlying water is removed carefully, refilling
again with cold distilled water. The stained cells can be observed with a conventional optical microscope.
- Klein method [34]
This “dry” argentophilic method reveals basically the cortical surface architecture, specially the argyrome that is an
important taxonomic feature of peritrichs and spirotrichs, among other ciliates.
Table 3
Klein method procedure.
Product
Silver nitrate (AgNO3)
Distilled water
Quantity
2g
100 ml
A drop of the sample is placed on a slide and fixed by leaving to air dry. A silver nitrate solution is then located on
top of the dried drop until the sample is totally covered and is left during 10-15 minutes exposing the wet slide at
sunlight or under a UV lamp. The excess of the staining solution is removed then and stained cells are washed slightly
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adding very carefully (to avoid detachment of cells) distilled water over the slide. Place a coverslip and observe directly
in a conventional microscope using the x40 to x100 objectives.
4.4
4.4.1
Other techniques
Image analysis
A procedure for a semi-automatic identification of the main protozoa and metazoan species present in the activated
sludge of wastewater treatment plants has been described by Ginoris et al. [35]. This procedure is based on both image
processing and multivariable statistical methodologies. This method provides recognition percentages above 80% for
the non-stalked organisms, although several misclassification problems were pointed out among certain groups. For the
stalked organisms, the individual recognition percentage was lower. However, when talking about main protozoa and
metazoan groups (flagellates, ciliates, sarcodines and metazoan) and protozoan ciliates (carnivorous, crawling,
swimming, sessile and not-ciliated) recognition attained good overall performance (around 95%).
4.4.2
Interactive guides
Several electronic guides have been published within the last decade. Eikelboom (2000) [36] published a manual
“Process Control of Activated Sludge Plants by Microscopic Investigation” together with a CD-ROM that provides a
powerful expert system for diagnosing and solving operational problems such as bulking and scum formation. It is
focused mainly on filamentous bacteria; however, it also includes a brief description of the most characteristic
biomarkers within the protist and microinvertebrate groups. On the other hand, Curds et al. (2008) [17] have recently
published an atlas of ciliated protozoa commonly found in aerobic sewage-treatment processes to help monitoring
treatments and assess plant performance. This is a multimedia, user-friendly guide for either specialists or nonspecialists and for both training and routine monitoring. This multimedia tool provides photographs and/or video clips
in vivo in addition to descriptions and line diagrams in order to mitigate the problem of recognising organisms observed
in fresh samples under the microscope. Video clips for species most commonly encountered in activated sludge, which
are considered of the greatest indicator value, are included. These video images show key behavioural patterns such as
swimming, feeding behaviour, etc, that are important for the temptative in vivo identification. Taxonomic descriptions
are also presented to emphasise differences between species known to occur in wastewater treatment, rather than
emphasising the features of systematic importance. It is possible to include in this tool the results of the sample
assessment to obtain a prediction of effluent quality.
Finally, other guides have also been published locally such as the Ferrer et al. (2009) [37] with a multimedia tool in
DVD as a basic application oriented to lab technicians in which the most relevant aspects to perform the analysis of
activated sludge are documented and illustrated. This tool has been elaborated from microscopic video clips from
different WWTP. This DVD contains the methodology for the activated sludge analysis, an interactive microbiota atlas
for protist and filamentous microorganisms including record forms for their identification and a tool to calculate biotic
indexes.
Acknowledgements The support by the Ministerio de Ciencia e Innovación through the project CGL2008-02310 and by the
Universidad Complutense de Madrid through the project PR1/08-15930-A is gratefully acknowledged.
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