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Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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 972 ©FORMATEX 2010 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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. ©FORMATEX 2010 973 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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. 2 974 b) Diversity of ciliates in activated sludge systems. a) Conventional WWTP and b) Advanced nutrient removal WWTP. ©FORMATEX 2010 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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 ©FORMATEX 2010 975 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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. 976 ©FORMATEX 2010 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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 ©FORMATEX 2010 977 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ 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. References [1] Metcalf-Eddy. Wastewater Engineering, Treatment and Reuse. Madrid: Mc. Graw-Hill; 2003. [2] Sezgin M, Jenkins D, Parker DS. A unified theory of filamentous activated sludgebulking. Journal Water Pollution Control. 1978;50:362-381. [3] Bitton G, Marshall KC. Adsoption of Microorganims to Surfaces. New York: Wiley Interscience; 1980. [4] Bitton G. Wastewater Microbiology. 3 rd ed. Hoboken, NJ:Wiley-Liss; 2005. [5] Curds CR. The flocculation of suspended matter by Paramecium caudatum. Journal of General Microbiology. 1963;33:357367. [6] Curds CR, Cockburn A. Protozoa in biological sewage treatment processes. II. Protozoa as indicators in the activated sludge process. A unified theory of filamentous activated sludgebulking. Water Research. 1970;4:237-249. [7] Curds CR. Protozoa in Sewage-Treatment Processes. Notes on Water Pollution. Ministry of Technology UK; 1968. [8] Grupo Bioindicación Sevilla. Manual Práctico para el Estudio de Grupos Bioindicadores en Fangos Activos. ed. Reed Business Information-Tecnología del Agua; 2008. [9] Madoni P. A sludge biotic index (SBI) for the evaluation of the biological performance of activated sludge plants based on the microfauna analysis. Water Research. 1994;28:67-75. [10] Salvadó H, Gracia MP, Amigó JM. Capability of ciliated protozoa as indicators of effluent quality in activated sludge plants. Water Research. 1995;29:1041-1050. [11] Martín-Cereceda M, Serrano S, Guinea A. A comparative study of ciliated protozoa communities in activated-sludge plants. FEMS Microbial Ecology. 1996;21:267-276. 978 ©FORMATEX 2010 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________ [12] Liu J, Yang M, Qi R, An W, Zhou J. Comparative study of protozoan communities in full-scale MWTPs in Beijing related to treatment processes. Water Research. 2008;42:1907-1918. [13] Pérez-Uz B, Arregui L, Calvo P, Salvadó H, Fernández N, Rodríguez E, Zornoza A, Serrano S. Efficiency of nitrogen removal and protist communities: the potential for introduction of novel biological index. Proceedings of the International Workshop on Integrated vision of urban and agro-industrial wastewater treatment, monitoring and reclamation: key role played by the Wastewater Treatment Plant. ISRIM/LIFE 2009:1-9. [14] Arévalo J, Moreno B, Pérez J, Gómez MA. Applicability of the sludge biotic index (SBI) for MBR activated sludge control. Journal of Hazardous Materials. 2009;167:784-789. [15] Madoni P, Davoli D, Chierici E. Comparative analysis of the activated sludge microfauna in several sewage treatment works. Water Research. 1993;27:1485-1491. [16] Madoni P. Role of protozoans and their indicator value in the activated sludge process. In: Madoni P, ed. Biological Approach to Sewage Treatment Process: Current Status and Perspectives. Perugia: Centro Luigi Bazzucchi; 1991:21-27. [17] Curds CR, Warren A, Salvado H, Roberts DM. An atlas of Ciliated Protozoa commonly found in aerobic sewage-treatment processes. An aid to monitor treatment -plant performance. London Natural History Museum 2008. Available at: http://ciliateguide.myspecies.info/ciliates-activated-sludge. Accessed 2008. [18] Norcross KL. Sequencing batch reactors-an overview. Water Science and Technology. 1992;26:2523-2526. [19] Shannon CE, Weaver W. The Matemathical Theory of Communication. Illinois: University of Illinois Press; 1963. [20] Salvadó H, Gracia MP. Determination of organic loading rate of activated sludge plants based on protozoan analysis. Water Research. 1993;27:891-895. [21] Arregui L, Muñoz-Fontela C, Guinea A, Serrano S. Direct visualization of the microtubular cytoskeleton of ciliated protozoa with a fluorescent taxoid. Journal of Eukaryotic Microbiology. 2002;49:312-318. [22] Lecke SB, Tasca T, Souto AA, De Carli GA. Trichomonas vaginalis: microtubule cytoskeleton distribution using fluorescent taxoid. Experimental Parasitology. 2002;102:113-116. [23] Page FC. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological Association, Ambleside; 1988. [24] Nassonova E, Smirnov A, Fahrni J, Pawlowski J. Barcoding Amoebae: Comparison of SSU, ITS and COI genes as tools for molecular identification of naked lobose amoebae. Protist. 2010;161:102-115. [25] Curds CR. An Illustrated Key to the British Freshwater Ciliated Protozoa Commonly Found in Activated Sludge.Water Pollution Research. 1969;12:1-90. [26] Foissner W, Berger H, Blatterer H, Kohmann F. Cyrtophorida, Oligotrichida, Hypotrichia, Colpodea. In: Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems. Informationsberchte des Bayer, Landesamt für Wasserwirtschaft 1/91. München; 1991:1-471. [27] Foissner W, Berger H, Kohmann F. Peritrichia, Heterotrichida, Odontostomatida. In: Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems. Informationsberchte des Bayer, Landesamt für Wasserwirtschaft 1/92. München; 1992:1502. [28] Foissner W, Berger H, Kohmann F. Hymenostomata, Prostomatida, Nassulida. In: Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems. Informationsberchte des Bayer, Landesamt für Wasserwirtschaft 1/94. München; 1994:1-548. [29] Foissner W, Berger H, Blatterer H, Kohmann F. Gymnostomatea, Loxodes, Suctoria. In: Taxonomische und ökologische Revision der Ciliaten des Saprobiensystems. Informationsberchte des Bayer, Landesamt für Wasserwirtschaft 1/95. München; 1995:1-540. [30] Fialkowska E, Fyda J, Pajdak-Stos A, Wiackowski K. Activated Sludge: Biology and Microscopic Analysis. Krakow: Impuls Publishers; 2005. (in polish). [31] Serrano S, Arregui L, Perez-Uz B, Calvo P, Guinea A. Guidelines for the Identification of Ciliates in Wastewater Treatment Plants. London: IWA Publishing; 2008. [32] Fernández-Galiano D. Silver impregnation of ciliated protozoa: procedure yielding good results with the pyridinated silver carbonate method. Transactions of the American Microscopical Society. 1976;95:557-560. [33] Fernández-Galiano D. The ammoniacal silver carbonate method as a general procedure in the study of protozoa of sewage (and other waters). Water Research.1994;28:495-496. [34] Klein BM. The “dry” silver method and its proper use. Journal of Protozoology. 1958;5:99-103. [35] Ginoris YP, Amaral AL, Nicolau A, Coelho MAZ, Ferreira EC. Development of an image analysis procedure for identifying protozoa and metazoa typical of activated sludge system. Water Research. 2007;41:2581-2589. [36] Eikelboom DH. Process Control of Activated Sludge Plants by Microscopic Investigation. London: IWA Publishing; 2000. [37] Ferrer C, Isac L, Serrano S, Arregui L, Perez-Uz B, Llopis JA, Claramonte J, Alonso S, GBS, Becares E, Alonso JL, Borras L. Biofac: La Bioindicacion en EDAR de Fangos Activos. Castellón: FACSA; 2009. (in spanish). ©FORMATEX 2010 979