Nova Biotechnol Chim (2017) 16(1): 54-60 DOI: 10.1515/nbec-2017-0008  Corresponding author: josef.trogl@ujep.cz Nova Biotechnologica et Chimica Comparison of bioindicator eukaryotes of activated sludge biocenoses on two water-treatment plants: a case study Farida Y. Achmadulinaa, Rustem K. Zakirova, Elena S. Balymovaa, Vera Denisovaa, Taťjána Brovdyováb,c, Josef Tröglb, and Martin Nerudab a Faculty of Food Technology, Kazan National Research Technological University, Karl-Marx-Str. 8, 420 015 Kazan, Russian Federation b Faculty of Environment, Jan Evangelista Purkyně University in Ústí nad Labem, Králova Výšina 3132/7, Ústí nad Labem, 400 96, Czech Republic c Faculty of Production Technologies and Management, Jan Evangelista Purkyně University in Ústí nad Labem, Pasteurova 3334/7, Ústí nad Labem, 400 96, Czech Republic Article info Article history: Received: 16th February 2017 Accepted: 28th June 2017 Keywords: Activated sludge Sludge microbial communities Waste-water treatment plant Biodiversity index Abstract Activated sludge biocenoses were compared on waste-water treatment plants in the city of Kazan, Russian Federation and the city of Teplice, Czech Republic. Based on Palia-Kovnatski index, Acanthamoeba in Kazan, Epistylis in Teplice, and Acanthamoeba and Centropyxis were dominant genera in both plants. The major subdominant generas identified were Arcella, Opercularia and Aspidisca. This indicates high nitrification ability, high water purification potential and matured activated sludge. Chemical composition of the waste-water was identified as the main factor determining the sludge biocenoses diversity. Higher sludge biodiversity (Shannon, Margalef, and Sorensen indexes) was found in Kazan corresponding to more concentrated inflow water.  University of SS. Cyril and Methodius in Trnava Introduction Currently, an assessment of the quality of wastewater incoming to and outgoing from a waste-water treatment plant (WWTP) is carried out mainly on the basis of chemical analyses. This brings accurate data with low detection limits however in longer time period required for analyses, without evaluation of the biological effect and providing limited information in conditions of multicomponent industrial effluents. The solution of the problem is in the complex use of chemical and biological monitoring of the treated waters and biocenoses of active sludge, since biological control methods allow an integral assessment of the conditions for the functioning of the bioagent (activated sludge) at any point in the biological treatment of wastewater. Nevertheless since their establishment the methods of bioindication have not developed significantly (Zhmur et al. 1997; Wanner et. al 2000). The influence of the biodiversity of a mixed population of microorganisms in water treatment processes on the composition of treated effluent is confirmed by earlier findings as well as by experience from operating wastewater treatment plants (Wanner et. al 2000; Ghanizadeh and Sarrafpour 2001; Seviour and Nielsen 2010; Zhmur 2003). However, the question remains open Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 07:37 UTC mailto:josef.trogl@ujep.cz Nova Biotechnol Chim (2017) 16(1): 54-60 55 whether the differences in the biological purification process can affect qualitative composition of the activated sludge biocenosis in wastewater. Recent works show the importance of knowledge of the composition and changes in the physiological state of microorganisms as indicator for assessing and predicting the effectiveness of biological oxidation process (Akhmadullina et al. 2016; Babko et al. 2014; Hreiz et al. 2015; Shchegolkova et al. 2016). Since this issue is not only of theoretical but also practical significance, we decided to carry out this case study and compare the activated sludge biocenoses of two wastewater-treatment plants (WWTP). The two localities chosen included the WWTP of the city of Kazan, Russian Federation, that is the industrial heart of autonomous Tatarstan Republic with ~1.2 mil. equivalent inhabitants. The second one, the city of Teplice, Czech Republic, is aSpa city with ~155 000 equivalent inhabitants. Both plant types show similarities in incoming water and sludge but differ in technology pattern. A comparison of the indicator organism profiles from two treatment plants by bio-indication (Miller et al. 2009) was carried out. The aim was the identification of dominant bioindicator microorganisms and evaluation of biodiversity (on the genus level) and similarities of sludge microbial communities based on diversity indices, especially Palia-Kovnatski. Table 1. Composition of wastewater in studied waste-water treatment plants. 1Purified water Abbreviations: BOD biochemical oxygen demand; COD chemical oxygen demand; WWTP waste-water treatment plant Experimental Sludge samples formed from similar urban waste- waters (for composition see Table 1) originated from two waste-water treatment plants. The first was localized in Kazan, Russian Federation, with traditional configuration of activation process (mechanical and biological wastewater treatment in aeration tanks that foresee the regeneration zone and aeration) (Fig. 1a). The second plant was by the spa city with low industry Teplice, Czech Republic, with nitrification-denitrification configuration (Fig. 1b). Samples were collected from the regeneration phase of activation process. Bioindication was carried out according to standards of Russian Federation (Bolotina 1987) using microscopes Micmed-5 (LOMO, Russia) and Lambda DN45 TH59F (Lambda Praha, Czech Republic). A small drop of liquid sludge on a microscopy glass slide was covered by cover glass and observed first at low magnification (eyepiece 10 or 15, lens 8), and then at large magnification (eyepiece 10 or 15 lens 40). Microscopic examination was carried out without removing the hand from the micrometer screw in order to be able to change focal lengths and consider the entire surface of the field. Identification of genera was carried out according to atlas of water and sludge microorganisms (Sládeček and Sládečková 1996, 1997). A genus-level of the sludge microbial community was investigated. Previous experiences of wastewater treatment aiming to control the activation process on-line showed that this level is sufficient for such purpose (Akhmadullina et al. 2016; Zakirov et al. 2013). All analyses were carried out in triplicate and evaluated using Microsoft Excel. Components (mg/L) WWTP Kazan WWTP Teplice Influent (mg/L) Efluent1 (mg/L) purification rate (%) Influent (mg/L) Efluent1 (mg/L) purification rate (%) BOD5 148.9 9.6 93.6 157.4 12.1 92.4 COD 576 68 88.2 335.1 50.2 85.1 Suspended solids 231.4 10.03 95.7 169.2 15.1 91.1 Total nitrogen 31.94 18.6 41.8 35.3 14.3 60.3 Total phosphorus 3.1 1.9 38.7 4.8 1.5 68.6 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 07:37 UTC Nova Biotechnol Chim (2017) 16(1): 54-60 56 Fig. 1. Scheme of biological treatment processes and sludge sampling sites: a) Waste water treatment plant Kazan; b) Waste water treatment plant Teplice. AS – activated sludge, W-W – waste water. Table 2. Classification of determined indicator organisms. Type Subtype Class Genus at the locality Kazan Teplice Sarcomastigophora Mastigophora Phymomastigophorea Astasia – Zoomastigophorea Bodo Bodo Sarcodina Lobosea Acanthamoeba Arcella Centropyxis Acanthamoeba Arcella Centropyxis Filosea Trinema Euglypha Trinema Euglypha Ciliophora Ciliata Oligohymenophora Paramecium – Kinetophragminophora Holophrya Chilodonella – Holophrya Chilodonella Colpidium Peritricha Epistylis Opercularia Vorticella Thuricola Epistylis Opercularia – – Polyhymenophora Aspidisca Aspidisca Suctoria Suctorida Podophrya Tokophrya – Tokophrya Nemathelminthes – Rotifera Lecane Cephalodella Rotaria Gastrotricha Callidina Gastrotricha – – Lecane Cephalodella Rotaria – – – Encentrum Philodina Nematodes Tobrilus Nematoda Tobrilus Nematoda Annelida – Oligochaeta Aelosoma Aelosoma Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 07:37 UTC Nova Biotechnol Chim (2017) 16(1): 54-60 57 Table 3. Indexes of biodiversity and similarities (Shitikov et al. 2003). WWTP Indexes Shannon Margalef Sorensen Kazan 3.88 3.42 0.77 Teplice 2.94 2.84 Results and Discussion Results of hydrobiological analyses are summarized in Table 2. These visual observations and calculated indexes of biodiversity and similarity (Table 3) show a highly similar profile of indicator organisms of the two studied sludge samples. Further they confirmed that composition of the waste water is the main determinant of the qualitative composition of microbial communities. The more rich genetic diversity of activated sludge treatment facilities in Kazan indicates to a more complex composition of treated wastewater. This was expected considering a half million inhabitants of the metropolis and industrial infrastructure development compared to Teplice with almost ten times less inhabitants. Systematization of data taking into account the specific conditions of existence of the identified biomarkers allowed us to classify active sludge of both WWTP as low-loaded, nitrifying, and performing the complete oxidation of pollutants. However, a comparison of the dominant aquatic biocenosis based on Palia-Kovnatsky index (D) (Table 4) confirmed the expected effect of arrangement of the biological treatment process on the composition of a mixed population of microorganisms (Table 4). Table 4. Palia-Kovnatsky indexes (D) of dominance in the plants at the two localities studied. Class Genus Palia-Kovnatsky index (D) at the locality Kazan Teplice Phymomastigophorea Astasia 1.6 – Zoomastigophorea Bodo 0.096 0.748 Lobosea Acanthamoeba 14.56 4.988 Arcella 3.2 4.239 Centropyxis 14.88 18.703 Filosea Trinema 3.072 0.935 Euglypha 14.24 17.706 Oligohymenophora Paramecium 1.632 – Kinetofragminophora Holophryа 1.152 0.623 Colpidium – 0.062 Chilodonella 0.32 0.623 Peritricha Epistylis 3.552 18.516 Vorticella 5.12 – Opercularia 4.416 8.354 Thuricola 0.096 – Polyhymenophora Aspidisca 7.168 2.431 Suctoria Podophrya 0.032 – Rotifera Gastrotricha 1.408 – Rotaria 0.064 0.249 Lecane 0.672 0.374 Cephalodella 1.28 0.249 Encentrum – 0.249 Nematodes Tobrilus 0.032 0.249 Nematoda 0.064 0.249 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 07:37 UTC http://medbiol.ru/medbiol/dog/000dadae.htm Nova Biotechnol Chim (2017) 16(1): 54-60 58 Fig. 2. Dominant genera of the Waste water treatment plant of the city of Kazan: а1, а2) Acanthamoeba; b) Centropyxis; c) Euglypha. Amorphous genera Acanthamoeba, Centropyxis, and Euglypha were dominant for the WWTP of the city of Kazan (D ≥10) (Fig. 2). Subdominant basic bio-indicators of water treatment process (D<10) were mainly ciliates – i.e. g. Aspidisca (Fig. 3). These are characteristic for nitrifying sludge and exhibit a wide range of tolerance to a variety of environmental factors. The presence of large ciliates of the genera Vorticella and Opercularia, also subdominant bioindicators, and high turbidity of the sludge liquid provide the evidence of deep regeneration of the activated sludge accompanied by its deflocculation (Fig. 3). In such conditions amorphous amoebea become competitive since they are capable of both saprozoic and holozoic type of nutrition (Zhmur 1997). Dominant bioindicator organisms found in WWTP Teplice, as expected, were the amorhous genera Centropyxis and Euglypha, typical for deep nitrifying sludge. As a confirmation of the latter fact is the dominance of peritrichous ciliates of the genus Epistylis, testifying to the high quality and good cleaning properties of nitrifying sludge. Among subdominants amorphous genera Acanthamoeba, Arcella, and ciliates Opercularia and Aspidisca prevail, characterizing ripe sludge. In this experimental case study, the relationships between the main technological factors (modification of the process) determining Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 07:37 UTC Nova Biotechnol Chim (2017) 16(1): 54-60 59 the functioning of the biological unit system and the biocenosis of the activated sludge were first investigated. The main impact of the results aims on the praxis of WWTP control (especially in the Czech Republic and Russian Federation, however, applicable worldwide) and its possible changes. Currently, an assessment of the quality of incoming and treated wastewater on WWTP is carried out, predominantly on the basis of chemical analysis data. In this regard, the informative nature of the chemical control of the treatment facilities is drastically reduced, causing an unrealistic reflection of the conditions for the functioning of the biocenosis of the activated sludge. This can result in low quality of the biologically treated effluents (Wanner et. al 2000; Balymova et al. 2011). Also toxic and poorly oxidizable components of industrial effluents might act in synergy on a mixed population of microorganisms and thus negatively affect the biological oxidation process. The complex use of chemical and biological monitoring of the treated waters and biocenosis of active sludge is possible way to resolve this problem, since biological control methods allow an integral assessment of the conditions for the functioning of the bioagent (activated sludge) at any point in the biological treatment of wastewater. In addition they are fast and they do not require sophisticated pricy equipment (except for optical microscope). Fig. 3. Deflocculating fibers of activated sludge (WWTP Kazan). Conclusions This study compared the qualitative composition of microbiota of two waste-water treatment plants. Composition of the waste-water was determined as the main factor determining composition of the sludge biocenoses. Higher sludge biodiversity (based on Shannon, Margalef, and Sorensen indexes) was found in Kazan corresponding to more concentrated inflow water. 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