The microfauna present in the activated sludge of a wastewater treatment plant is mainly composed by bacteria, protozoa and metazoa. The protozoan species are quite sensitive to physical, chemical and operational processes making them, thus, precious indicators of the state of the plant. Several authors already established relationships between the predominance of certain species or group and some parameters of the plant, such as the biotic indices namely the Sludge Biotic Index. All the above-mentioned procedures demand the identification, classification and quantification of the different species. Normally this is done manually, which implies both time and expertise. In the present work a semi-automatic protozoan recognition procedure by means of image analysis is attempted. The program built for this purpose (ProtoRec v.3) was also used to study the evolution of the microfauna during transient operation times (stoppage and re-run). The results were rather satisfactory in terms of protozoa recognition and the survey of the transient phase allowed verifying the aging and degradation of the microfauna by means of the different predominant species

Amaral, A. L.

Ferreira, E. C.

Mota, M.

Motta, Maurício da

Pons, M. N.

Vivier, H.

Ferreira, Eugénio C.

Universidade do Minho: RepositoriUM

SURVEY OF A WASTEWATER TREATMENT PLANT MICROFAUNA BY IMAGE ANALYSIS  A.L. Amaral1, M. da Motta2,3, M.N. Pons2, H. Vivier2, M. Mota1, E.C. Ferreira1*  1 Centro de Engenharia Biológica - IBQF, Universidade do Minho, 4710-057 Braga, Portugal 2 Laboratoire des Sciences du Génie Chimique, CNRS-ENSIC-INPL, 1 rue Grandville, BP 451, F-54001 Nancy cedex, France 3 UFPE, Departamento de Engenharia Química, Av. Prof. Artur de Sá, s/n - Cidade Universitária - Recife 50740-521 Pernambuco Brasil (mottas@npd.ufpe.br), tel. ++ 55 (0)81 3 271 8238 fax ++ 55(0)81 3 271 0095   Abstract  The microfauna present in the activated sludge of a wastewater treatment plant is mainly composed by bacteria, protozoa and metazoa. The protozoan species are quite sensitive to physical, chemical and operational processes making them, thus, precious indicators of the state of the plant. Several authors already established relationships between the predominance of certain species or group and some parameters of the plant, such as the biotic indices namely the Sludge Biotic Index. All the above-mentioned procedures demand the identification, classification and quantification of the different species. Normally this is done manually, which implies both time and expertise. In the present work a semi-automatic protozoan recognition procedure by means of image analysis is attempted. The program built for this purpose (ProtoRec v.3) was also used to study the evolution of the microfauna during transient operation times (stoppage and re-run). The results were rather satisfactory in terms of protozoa recognition and the survey of the transient phase allowed verifying the aging and degradation of the microfauna by means of the different predominant species.   1. Introduction  In the aeration tank of a wastewater treatment plant bacteria agglomerate generally in flocs. Attached to these flocs and swimming in the surrounding liquid one can find also bacteria, protozoa and metazoa may also be found. Interactions between these groups include predation (I), competition (II) and cannibalism (III) relationships, as seen in figure 1.   Figure 1 – Relationships between the different microorganism species in the aeration tank (Canler et al., 1999)   The protozoan presence is fundamental for the good performance of the plant. As well as decreasing the turbidity of the final effluent they also stimulate the bacterial growth, promote the flocculation of the sludge and some species are responsible for the elimination of the pollution (Gerardi et al., 1995; Curds et al., 1968). 1137 T5 – Biochemical Engineering After bacteria, protozoa are the most important microorganisms in wastewater treatment. Their high sensibility to the variations of their environment (substrate and dissolved oxygen concentration, toxics presence, etc) makes them excellent indicators of the plant state (Degrémont, 1989).  The importance of the protozoan species, mainly bacteriophages, can be summarized as follows: by grazing the free bacteria they decrease the turbidity of the effluent as well as the Biochemical Oxygen Demand (BOD), the dry mass percentage and the pathogenic bacteria (95% and 50% respectively with and without their presence, accordingly to Gerardi et al., 1995); their bacteriophage regime stimulates the bacterial growth and the development of new flocs. One final remark, although subject to controversy is that the protozoa can eliminate pollution in terms of organic matter.  Protozoan species can be classified in terms of flagellates, ciliates and amoebae. The ciliates, present in higher concentrations, can be also classified accordingly to their feeding regime into bacteriophages and carnivores. They are also classified accordingly to their mobility in free swimming, crawling and stalked ciliates.  Several authors studied the different ciliate species present in an aeration tank in order to identify them (Curds and Cockburn, 1970a; Sasahara and Ogawa, 1983), as well as the correlation between the plants performance and the abundance of certain species (Curds and Cockburn, 1970b; Madoni, 1994a; Nicolau et al., 1997). Table I summarizes the relationships between each predominant protozoan group and plant efficiency, as well as probable problem causes.  Table 1 – Relationships between protozoa and plant efficiency (Madoni, 1994a)  Predominance Efficiency Probable cause Small flagellates Bad Low oxygenated sludge; high organic load; presence of fermentescible substances. Small swimming ciliates  (< 50 µm) Mediocre Low contact time, low oxygenated sludge.  Large swimming ciliates  (> 50 µm) Mediocre High organic load. Crawling ciliates Good  Stalked + crawling ciliates  Good  Stalked ciliates Decreasing Transient phenomena (discontinuous load; recent sludge removal).  Small amoebae and flagellates Bad High organic load with low biodegradability. Testate Amoebae Good Low organic load; good nitrification.  The sludge colonization starts with the bacteria, flagellates and free-swimming ciliates that are normally present in the incoming effluent. As the sludge grows old the crawling and the stalked ciliates begin to settle. Contrarily to the first species these two last ones return by the recirculation channel. The reduction of the bacteria is due primarily to the bacteriophage regime of the protozoa species. Figure 2 summarizes the evolution of the different microorganisms as function of the sludge age.  1138 A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira Survey of a Wastewater Treatment Plant Microfauna by Image Analysis …   Figure 2 – Evolution of each microorganisms group as a function of sludge age  (Canler et al., 1999).  The Sludge Biotic Index (SBI) proposed by Madoni (1994b) uses the plant ecosystem sensibility on the physical, chemical and operational conditions and is based on the different sensitivities of each protozoan group to the above-mentioned parameters. In order to quantify the different protozoan species identification and classification steps, are needed in the first place.   Normally the identification and quantification of each species is done by visual inspection and manual counting, which requires both time and expertise. In the present work, a semi-automatic program (ProtoRec) was developed aiming to identify by means of morphological descriptors the different protozoan species (Amaral et al., 1999; Da Motta et al., 2001).   2. Materials and Methods  Activated sludge samples were gathered from an aeration tank of a wastewater treatment plant in Braga. A drop of each sample was mounted on a microscope slide and the image acquisition was done by direct illumination without the addition of any dyes. From each sample images of 50 individuals were acquired.  The image acquisition system consists of an Axioskop epi-fluorescence microscope (Zeiss, Oberkochen), connected to a CCD AVC D5CE camera (Sony, Tokyo). The signal is then treated by a CMA-D5CE adaptor (Sony, Tokyo) and is finally digitized by a Meteor 2 frame grabber (Matrox, Dorval).  A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira 1139 T5 – Biochemical Engineering   Figure 3 – Image acquisition system  Figure 4 shows some of the main steps of the ProtoRec v.3 program. First, the original image (256 gray scales) is pre-treated to enhance the protozoon contour. Secondly the regions of interest are traced by the user and the resulting image is consequently thresholded. Small debris are then removed and the protozoa are labeled. Finally the morphological parameters of each protozoan are determined. The identification step is obtained by comparison of these values to a protozoan database.    Original image  Pre-treated image   Regions of Interest  Recovered protozoa  Binary image   Final labeled image  Figure 4 – Main steps of ProtoRec v 3   The morphological parameters determined by ProtoRec v.3 are the following: Area, Equivalent Diameter, Perimeter and Feret Shape; Eccentricity (Glasbey, 1994); Length, Shape factor, Width, Compacity, Convexity and Solidity (Russ, 1994); Robustness, Largest Concavity Index, Concavity Ratio and Euclidean Distance Fractal Dimension (Pons, 1999); Mean Width and Mean Width / Width. These parameters are determined for both the entire protozoan and the protozoan body. The following parameters are also determined: Body Perimeter / Perimeter, Stalk Mean Width, Stalk Mean Width / Body Mean Width.   1140 A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira Survey of a Wastewater Treatment Plant Microfauna by Image Analysis … 3. Results  During the Winter of 2000/01, Braga went trough heavy rain falls, which gave rise to several problems in the wastewater treatment plant, caused by the high loading rates and sand build up leading to a cleaning stoppage. During this time the microfauna of the plant was surveyed starting from 19/03/01 when the plant was yet functioning normally. At 20/03/01 the incoming effluent was suspended (both recirculation and aeration were kept running), at 26/03/01 (last day of the survey) the recirculation was stopped and in the next day the aeration was also stopped.  The results obtained from this survey allowed for the manual analysis of the microfauna of the aeration tanks in a transient phase of the plant.  3.1. Manual Analysis of the Microfauna  As suggested by figure 2, the aging process of the sludge can be monitored by the evolution of the protozoan and metazoan groups present. First, a reduction of the swimming ciliates (Trachelophyllum) was observed due to their removal in the settling tanks. This phase was followed by the increase in the crawling ciliates (feeding on floc surface bacteria). As this substrate decreased the number of the crawling ciliates was also reduced. Stalked ciliates that initially had decreased their number increased again, mainly by the presence of Opercularia, which is resistant to stress conditions such as oxygen or nutrient limitation. Analyzing table I it is clear that the plant suffered a breakdown in its efficiency. Given these results it might be concluded that up to 3 days of stopping influent feeding the incoming effluent suspension (keeping both recirculation and aeration running) the plant could be easily re-started.  19/321/323/325/3StalkedCrawlingCarnivoreTrachellophylumAmoebaeMetazoan0102030405060708090100Percentage (%)Time (days) Figure 5 – Evolution of the different protozoan and metazoan groups as a function of time.   Figure 6 allows registering the reduction of the microfauna diversity: from 11 different species observed in day 1 to only 5 at the end of the survey.  Initially there is a strong predominance of the swimming ciliate Trachelophyllum with 45 % of the total protozoan and metazoan species. This ciliate feeds on bacteria and small flagellates and is present at any loading rate, but according to Canler et al. (1999) its A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira 1141 T5 – Biochemical Engineering concentration increases with higher loading rates. Its predominance is related with a mediocre depurative efficiency and is an indicator of transient phenomena such as overloads.   At day 3 Trochilia predominated, which is an indicator of a good effluent quality and is usually present in plants in steady state operation (Canler et al., 1999). Its presence may indicate the catabolic process where bacteria consume their energetic reserves, producing mineral salts such as the nitrates.  Finally a strong predominance of Opercularia was encountered. This protozoan is resistant to stress conditions such as the presence of certain toxics (mineral salts, heavy metals, etc), acidity and lack of aeration, which reflects mediocre to sufficient efficiencies and high concentrations of NH4+ in the treated effluent.   This survey allows confirming the reduction in the depurative efficiency after 6 days without fresh effluent. The organic matters catabolism accounts for the probable presence of NH4+ in the final effluent.  19/03/0120/03/0121/03/0122/03/0123/03/0124/03/0125/03/0126/03/010.00.10.20.30.40.50.60.70.80.91.0AmoebaeProrodonZoothamniumLitonotusTrithigmostomaEpystilisCharchesiumNematodaRotifersTrachellophylumVorticellaAspidisca cicadaTrochilliaOpercularia Figure 6 – Evolution of the protozoan and metazoan species present as a function of time.    3.2. Species Recognition with ProtoRec v.3   The program ProtoRec v.3 was used to determine the above-mentioned morphological parameters. These parameters were furthermore treated with the multivariate statistical technique Discriminant Analysis (Einax et al., 1997). This technique has two major advantages: it decreases the number of relevant parameters (calculated as a linear combination of the initial parameters) and allows for a better separation of different groups of objects. A total number of 50 individuals of each protozoon species was used in the discriminant analysis (except for Trochilia with only 37 individuals) and further 50 (of each specie) were used for the validation process. In the latter process, however, several species did not possess this number of individuals, which was the case of Carchesium (35), Digononta (17), Nematoda (7), Aelosoma (16) and Suctoria and Trochilia for which an estimate from the original values had to be made.  1142 A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira Survey of a Wastewater Treatment Plant Microfauna by Image Analysis …  Although Nematoda (Class), Suctoria (Sub-class), Monogononta (Order) and Digononta (Order) are not species they are referred as species in table II for simplification reasons.  The results obtained for the validation are represented in table II.  Table II – Recognition percentages for each species.    % REC.  NEMATODA 100 % SUCTORIA 98 % * TROCHILIA 95 % * LITONOTUS 90 %  Very Good  PERANEMA 88 % ARCELLA 86 % TRACHELLOPHYLLUM 86 % EUPLOTES 84 % AELOSOMA 81 % EUGLYPHA 80 % ASPIDISCA CICADA 78 % VORTICELLA AQUADULCIS 78 % MONOGONONTA 76 %  Good TRITHIGMOSTOMA 74 % DIGONONTA 71 % VORTICELLA MICROSTOMA 70 % VORTICELLA CONVALLARIA 66 % EPISTYLIS 56 % ZOOTHAMNIUM 52 %  Reasonble CARCHESIUM 43 % OPERCULARIA 42 %  Poor * Estimates  The average recognition value for each species was 75%.  The results obtained for the classification in the main protozoan and metazoan groups are in table III. Within the ciliate group it is also useful to perform another classification (table IV).  Table III – Recognition percentages for each group. Table IV – Recognition percentages for each ciliate class.   % REC CILIATES 94 % FLAGELATES 88 % METAZOAN 86 % TESTATE AMOEBAE 83 %    % REC CARNIVORES 94 % CRAWLING 92 % STALKED 90 % FREE SWIMMI G N 86 %   The average recognition value for each group was 92% and for each class within ciliates 91%.  A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira 1143 T5 – Biochemical Engineering 4. Conclusions  The results obtained with ProtoRec can be regarded as quite promising. The average recognition value for each species was 75%, for each group 92% and for each class within ciliates 91%.  The wastewater treatment plant survey allowed for the identification of initially free swimming ciliates (Trachelophyllum), followed by crawling ciliates (Trochillia) and finally stalked ciliates (Opercularia) as well as some metazoan species.   Bibliography  Amaral A.L., Baptiste C., Pons M.N., Nicolau A., Lima N., Ferreira E.C., Mota M., Vivier H., (1999) Semi-automated recognition of protozoa by image analysis, Biotechnology Techniques, 13, p. 111-118. Canler J.-P., Perret J.-M., Duchène P., Cotteux E. (1999) Aide au diagnostic des stations d'épuration par l'observation microscopique des boues activées, Cemagref Editions, Paris. Curds C.R., Cockburn A., Vandike J.M. (1968) An experimental study of the role of the ciliated protozoa in the activated-sludge process, Water Pollution Control, 67, p. 312-329  Curds C.R., Cockburn A. (1970a) Protozoa in biological sewage-treatment process: I. a survey of the protozoan fauna of British percolating filters and activated-sludge plants, Water Research, 4, p. 225-236. Curds C.R., Cockburn A. (1970b) Protozoa in biological sewage-treatment process – II. protozoa as indicator in the activated-sludge process, Water Research, 4, p. 237-249. Gerardi M.H., Horsfall F.L. (1995) Wastewater Biology: The microlife, A Special Publication, Water Enviroment Federation, Virginie. Da Motta M., Pons M.-N., Vivier H., Amaral A. L., Ferreira E. C., Roche N., Mota M. (2001) Study of protozoa population in wastewater treatment plants by image analysis, Brazilian Journal of Chemical Engineering , 18 (1), p. 103 -111 Degrémont (1989) Mémento Technique de L'eau, Vol. 1, Lavoisier, Paris. Einax J.W., Zwnziger H.W., Geiss S. (1997). Chemometrics in environmental analysis, VCH - Wiley company, Weinheim. Glasbey C.A. (1994) Image analysis for the biological sciences, John Wiley & Sons, Chichester. Madoni P. (1994a) La microfauna nell'analisi di qualità biologica dei fanghi attivi, AGAC di Reggio Emilia - Universita degli Studi di Parma, Parma. Madoni P. (1994b) A Sludge Biotic Index (SBI) for the evaluation of the biological performance of activated sludge plants based on the microfauna analysis, Water Research, 28 (1), p. 67-75. Nicolau A., Lima N., Mota M., Madoni P. (1997) Os protozoarios como indicadores da qualidade biologica de lamas activadas, Boletim de Biotecnologia, 56, p.14-19.  Pons M.-N. (1999) Biomass quantification by image analysis, Advances in Biochemical Engineering / Biotechnology, 66, p. 133-184. Russ C.R. (1994) The image processing handbook, CRC Press, Boca Raton. Sasahara T., Ogawa T. (1983) Treatment of brewery effluent. part IV: protozoa and metazoa found in activated sludge process for brewery effluent, Monatsschrift für Brauwissenschaft, 11, p. 433-448.  1144 A. Amaral, M. Motta, M.N. Pons, H. Vivier, M. Mota, E.C. Ferreira 

Survey of a wastewater treatment plant microfauna by image analysis

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Survey of a wastewater treatment plant microfauna by image analysis

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