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Biofilm characterization of several wastewater treatment plants with rotating biological contactors in Madrid (Spain)
~
Pergamon
Wal. Sci. T~ch. Vol. 37. No. 4-5. pp. 215-218. 1998. (l) 1998 IAWQ. Published by Elsevier Science LId Printed In Great Britain. 0273-1223198 S19'00 + 0'00
PH: S0273-1223(98)00 I09-7
BIOFILM CHARACTERIZATION OF SEVERAL WASTEWATER TREATMENT PLANTS WITH ROTATING BIOLOGICAL CONTACTORS IN MADRID (SPAIN) B. Perez-Uz, c. Franco, M. Martin-Cereceda, L. Arregui, I. Campos, S. Serrano, A. Guinea and D. Fernandez-Galiano Departamento de Microbiologla 1//. Facultad de Ciencias BiolOgicas. Universidad Complutense. 28040 Madrid. Spain
ABSTRAcr Ciliate communities .were studied in the biofilms of three plants with rotating biological contactors (RBCs) over the period of one year. The aims of this study were: (i) to characterize ciliates to species level; (ii) to delennine their abundance and spatial variation through the biological system; and (iii) to relate the spatial segregation and richness of ciliate species to plant performance. A number of species ranging from 33 to 67 were identified at the different plants. The overall best represented groups were peritrichs followed by scuticociliales (Las Matas). cyrtophorids (Boadilla) or hypotrichs (Camarma). Comparison of RBCs and activated sludge communities indicated that even though both were mainly constituted by peritrichs. differences in groups and species composilion were observed. © 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Biofilm; ciliates; peritrichs; RBC compartmentalization; wastewater. INTRODucrlON Ciliated protozoa are important members of the communities involved in the removal of organic matter and bacteria in biological wastewater treatment plants (Curds, 1993). Comparative studies on the occurrence of ciliates in several RBC systems are not common (Madoni and Ghetti, 1981; Kinner et aI.• 1989; Chung and Strom. 1991). Some of this work has been only on the colonization process on a pilot plant scale (Kinner and Curds, 1987). Other authors have provided lists of identified species comparing activated sludge and RBC systems (Madoni and Ghetti, 1981). However, generally no emphasis has been placed on the compartmentalization of the RBC process. This feature allows particular communities to develop at the same time in the different contactors along an RBC process train. The major objective of this study was to describe the ciliate communities found in full scale RBC sewage treatment plants located in Madrid (Spain) from this point of view. Results on diversity and abundance as related to spatial variability over a one year period are presented. Differences between these communities and those in activated sludge processes are JWST 37:./5-U
215
B. PEREZ-UZ et al.
216
also discussed. This research is part of a project to integrate biological and physical-chemical variables and to define the use of ciliates as indicators of the RBC process performance. METHODS Three plants with RBC systems at different locations in the Madrid area (Las Matas, Camarma and Boadilla) were selected for this study. Samples from both biofilm (23.75 cm 2) and water (I I) were taken at 3 or 4 points along the RBC system depending on the plant design. Biofilm samples were delimited by using the sample bottle mouth as a corer, then scraped from the RBC disk and mixed with 0.45 J1m filtered water from the same point to bring the total volume up to 20 ml. Triplicate ciliate counts were done on 25 J11 of this biofrlm suspension. Total solids (TS) and volatile solids (VS) were also measured on replicate samples of biofilm. and the number of individuals were referenced to grams of dry biofilm. Physical-chemical analysis (pH. temp. DO. NOf-N. NOf-N. NH 3-N. P043-, BODs. SS) on water samples were done according to Standard Methods (APHA. 1989). Ciliate identifications were done in vivo with phase contrast microscopy and after silver carbonate (Fernc1ndez-Galiano. 1994) and protargol (Wilbert, 1975) staining methods. These techniques were used to reveal infraciliature characters essential for identifying species. RESULTS AND DISCUSSION The number of ciliate species depended on the plant and ranged from 33 to 67. Table I shows the most representative species for each plant placed into their higher taxonomic groups (Order). Group abundances were obtained as percentages of the populations for the entire process. Peritrich ciliates were the dominant group in all plants with approximately 80% of the total abundance. This can be explained because peritrichs are attached ciliates. well adapted to colonize biofilms using their stalks for attachment. Table I. Ciliate species· in the three RBC systems studied
Las Matas
Peritrichs
Vorlf~/Ia convaJ/arla Epistylis spp.
QrcMsi_sp. ZooIhamn/lI",
Hypotrichs Cyrtophorids
Hymenostomes Scuticociliates
Pleurostomids Prostomids Suctorians Heterotricha
sp.
AspidtSCD cicada OIi/odoMlla llne/nala Trochilia ",i""la Parameciu", CQlldotu", Paramecill", aurrlia Deriotrlcha tmnqullla Cinelochllll", margarllaMI", Utonotru '-lla Ac/nerla uncinola
Camanna
Boadilla Vorlf~lla convaJ/arla Epistylisplicati/is Zoothamnillm procmlU Opm:IIlarla c:oarcIala AspidiSCD cicada Trochilia ",iflllla ChilodoMlla uneinola Parameci_ aurelia Paramec/II", CQlldatu",
Cinelochllll", margarllaMI'" Deriotrlcha sp. LJtOltOlrU
Opm:II/arla artiClilaJa OpefCIIltria COt11'Cfa/a AspidiSCD cicada ChilodoMlla II1Icinola Deriostoma ClImpy/a TetmJrymena wt!omI/S Parameciu", caudotu", Uronema "igrlCtlfl.f
'-'/a
AciMrla uncinola
TrlI1l)'mlQ comprrSSllllf EncJte/yontor'pha vmn/C11Iarl.J Ak/opflS sp.
• Percentage considered higher than I%. Species listed in decreasing order ofabundance.
Biofilm characterization of several wastewater treatment plants
217
Different abundances of total ciliates were found along the RBC system in the 3 plants. In Las Matas and Boadilla. the second sampling point (corresponding to an intermediate stage of biological treatment) had the most populated biofilm (1.60 x 106 cil g-1 TS and 1.72 x 106 cil g'\ TS respectively). However, in Cam anna. the highest abundance of ciliates was at the fourth sampling point (last stage in the process) with an average number of 4.87 x 106 cil g-) TS. In this case, the great increase in abundance was due to the presence of Opercu/aria articulata and Aspidisca cicada in large numbers on just 4 sampling dates. oI,-r-----./
La, Mata'
.,/
80•
Penulchs
~f.iii'i~:;.cr Pentnchs
-"
60
Hl')lOIl"lOhs Cyrtophotlds
H)'!Il
40· 20 -
5cutJCOCIh... PI''''O.tMUds
Prostonl.ds Suetonans
.,/
/
~
Hvpotnclls
Cynophonds
-.G~~lv~ s~: C7''/ P1ewostomJds ,/ ProstMud. / . SuetonlJl.
oL._ -----..t/ Heteroo.oh.
H,1CrOtnchs
I 2 3 //
Camarma /
100/ 8060•
40· 20-
40'
Q -../. ./ SuetonlJl. o~ Heter_ebs I 2 3 4
Figure I. Abundance (%) and distribution of ciliates groups at different sampling points in the
RBe plants.
The distribution of groups and their relative abundances through the system are shown in Fig. I. Results indicated a variation in the abundance groups depending on the stage of biological treatment. Las Matas and Boadilla had similar distribution patterns. In both plants, peritrichs were the most representative ciliates in all stages, having the lowest abundance at the last sampling point. The most abundanct peritrich species were Vorticella convallaria (Las Matas) and Epistylis spp. (Boadilla). The abundance of these species decreased towards the last stage where other species were favoured (i.e. Zoothamnium spp.). Also in both cases. pleurostomids reached their highest abundance in the last stage (mainly the species Litonotus lamella) while hymenostomes were most abundant in the first stage. The main difference between Las Matas and Boadilla concerned scuticociliates, which behaved oppositely throughout the system. This was due to the occurrence of the species Cinetochilum margaritaceum that appeared with the highest abundance in the last stage of Las Matas while at Boadilla it was mostly present in the first stage. Heterotrichs were present in amounts higher than I % only in Las Matas. Camarma showed a different spatial distribution and abundance of groups compared to the other plants (Fig. I). Peritrichs (mainly Opercu/aria articulata) were the most abundant ciliates but only in the last stage of the process. Previous stages of treatment showed as hymenostomes dominant organisms at the beginning of the process and hypotrichs in the intermediate stages. Other groups well represented in the first contactor were scuticociliates (Uronema nigricans and Dexiotricha tranquilla), prostomids (Trimyema compressum) and suctorians (Enche/yomorpha vermicu/aris). The last two did not appear in significant amounts in the other plants. A gradual decrease of
218
B. PEREZ-UZ et al.
suctorians. scuticociliates, hymenostomes and prostomids was observed along the treatment stages in this plant. Good performance was found at Las Matas and Boadilla with an efficient BODs removal through the RBCs (55-70%). Both plants showed similar spatial distribution of ciliate communities. This could indicate that these communities might be associated with an efficient purification process. Nevertheless, Camarma showed lower diversity of ciliates with the dominance of only a few species. This may be related to poor performance, since the BODs removal by the biological process was only 20%. The occurrence of high numbers of certain species -Trimyema compressum and Enchelyomorpha vermicularis - which are especially adapted to poorly oxygenated conditions also suports this hypothesis. A previous study (Martfn-Cereceda et aI., 1996) in to activated sludge plants also located in Madrid (Spain), has allowed us to compare ciliate communities from both types of wastewater treatment plant. Peritrichs are the most representative ciliates in these processes. Species like Paramecium caudatum. P. aurelia, P. putrinum, Metopus sp., Brachonella spiralis, Trimyema compressum, Colpoda aspera and Enchelyomorpha vermicularis reported by other authors (Madoni and Ghetti, 1981; Augustin and Foissner, 1992) in activated sludge have only been found by us in RBCs. CONCLUSIONS RBC plants with high BODs removal efficiencies had richer populations of ciliate species than plants with poor treatment efficiency. Peritrichs made up the highest proportion of ciliates in these ecosystems. The careful characterization of ciliate communities inhabiting RBC biofilms performed in this research improves the knowledge of the role of these microorganisms in wastewater treatment plants. ACKNOWLEDGEMENTS This work was financially supported by C.A.M. (Project 1+0 0125/94). REFERENCES Augustin. H. and Foissner. W. (1992). Morphologie und Clkologie einiger Ciliaten (Protozoa: Ciliophora) aus dem Belebtschlamm. Arch. Protistenkd.• 141(4), 243-283. Chung. J. and Strom, P. (1991). Microbiological study of ten New Jersey rotating biological contactor wastewater treatment plants. Res. J. Wat. Pollut. Control. Fed.• 63(1),35-43. Curds. C. R. (1993). Interactions Protozoaires·Bacteries dans les methodes aerobies de lraitement des eaux usees. Ann. Bioi.• 32(1 ).1-12. Kinner. N. E. and Curds, C. R. (1987). Development of protozoan and metazoan communities in rotating biological contactor biolilms. Wat. Res., 21(4),481-490. Kinner, N., Curds. C. R. and Meeker, L. D. (1989). Protozoa and Metazoa as indicators of effluent quality in rotating biological contactors. Wat. Sci. Tech .• 20(11112).199-204. Fernandez·Galiano. D. (1994) The ammoniacal silver carbonate method as a general procedure in the study of protozoa from sewage (and other) waters. Wat. Res., 28(2). 495-496. Madoni, P. and Ghelli. P. F. (1981). The structure of ciliated protozoa communities in biological sewage·treatment plants. Hydrobiologia. 83, 207-21 S. Manln·Cereceda. M., Serrano, S. and Guinea. A. (1996). A comparative study of ciliated protozoa communities in activated· sludge plants. FEMS Microbiol. £Col.• 21. 267-276. Standard Methods for the Examination of Water Quality and Wastewater (1989). 17th edn, American Public Health Association/American Water Works AssociationlWater Environment Federation, Washinghton DC, USA. Wilbert. N. (1975). Eine verbesserte Technik der Protargolimpragnation fUr Ciliaten. Mikrokosmos, 64. 171-179.
Pergamon
Wal. Sci. T~ch. Vol. 37. No. 4-5. pp. 215-218. 1998. (l) 1998 IAWQ. Published by Elsevier Science LId Printed In Great Britain. 0273-1223198 S19'00 + 0'00
PH: S0273-1223(98)00 I09-7
BIOFILM CHARACTERIZATION OF SEVERAL WASTEWATER TREATMENT PLANTS WITH ROTATING BIOLOGICAL CONTACTORS IN MADRID (SPAIN) B. Perez-Uz, c. Franco, M. Martin-Cereceda, L. Arregui, I. Campos, S. Serrano, A. Guinea and D. Fernandez-Galiano Departamento de Microbiologla 1//. Facultad de Ciencias BiolOgicas. Universidad Complutense. 28040 Madrid. Spain
ABSTRAcr Ciliate communities .were studied in the biofilms of three plants with rotating biological contactors (RBCs) over the period of one year. The aims of this study were: (i) to characterize ciliates to species level; (ii) to delennine their abundance and spatial variation through the biological system; and (iii) to relate the spatial segregation and richness of ciliate species to plant performance. A number of species ranging from 33 to 67 were identified at the different plants. The overall best represented groups were peritrichs followed by scuticociliales (Las Matas). cyrtophorids (Boadilla) or hypotrichs (Camarma). Comparison of RBCs and activated sludge communities indicated that even though both were mainly constituted by peritrichs. differences in groups and species composilion were observed. © 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Biofilm; ciliates; peritrichs; RBC compartmentalization; wastewater. INTRODucrlON Ciliated protozoa are important members of the communities involved in the removal of organic matter and bacteria in biological wastewater treatment plants (Curds, 1993). Comparative studies on the occurrence of ciliates in several RBC systems are not common (Madoni and Ghetti, 1981; Kinner et aI.• 1989; Chung and Strom. 1991). Some of this work has been only on the colonization process on a pilot plant scale (Kinner and Curds, 1987). Other authors have provided lists of identified species comparing activated sludge and RBC systems (Madoni and Ghetti, 1981). However, generally no emphasis has been placed on the compartmentalization of the RBC process. This feature allows particular communities to develop at the same time in the different contactors along an RBC process train. The major objective of this study was to describe the ciliate communities found in full scale RBC sewage treatment plants located in Madrid (Spain) from this point of view. Results on diversity and abundance as related to spatial variability over a one year period are presented. Differences between these communities and those in activated sludge processes are JWST 37:./5-U
215
B. PEREZ-UZ et al.
216
also discussed. This research is part of a project to integrate biological and physical-chemical variables and to define the use of ciliates as indicators of the RBC process performance. METHODS Three plants with RBC systems at different locations in the Madrid area (Las Matas, Camarma and Boadilla) were selected for this study. Samples from both biofilm (23.75 cm 2) and water (I I) were taken at 3 or 4 points along the RBC system depending on the plant design. Biofilm samples were delimited by using the sample bottle mouth as a corer, then scraped from the RBC disk and mixed with 0.45 J1m filtered water from the same point to bring the total volume up to 20 ml. Triplicate ciliate counts were done on 25 J11 of this biofrlm suspension. Total solids (TS) and volatile solids (VS) were also measured on replicate samples of biofilm. and the number of individuals were referenced to grams of dry biofilm. Physical-chemical analysis (pH. temp. DO. NOf-N. NOf-N. NH 3-N. P043-, BODs. SS) on water samples were done according to Standard Methods (APHA. 1989). Ciliate identifications were done in vivo with phase contrast microscopy and after silver carbonate (Fernc1ndez-Galiano. 1994) and protargol (Wilbert, 1975) staining methods. These techniques were used to reveal infraciliature characters essential for identifying species. RESULTS AND DISCUSSION The number of ciliate species depended on the plant and ranged from 33 to 67. Table I shows the most representative species for each plant placed into their higher taxonomic groups (Order). Group abundances were obtained as percentages of the populations for the entire process. Peritrich ciliates were the dominant group in all plants with approximately 80% of the total abundance. This can be explained because peritrichs are attached ciliates. well adapted to colonize biofilms using their stalks for attachment. Table I. Ciliate species· in the three RBC systems studied
Las Matas
Peritrichs
Vorlf~/Ia convaJ/arla Epistylis spp.
QrcMsi_sp. ZooIhamn/lI",
Hypotrichs Cyrtophorids
Hymenostomes Scuticociliates
Pleurostomids Prostomids Suctorians Heterotricha
sp.
AspidtSCD cicada OIi/odoMlla llne/nala Trochilia ",i""la Parameciu", CQlldotu", Paramecill", aurrlia Deriotrlcha tmnqullla Cinelochllll", margarllaMI", Utonotru '-lla Ac/nerla uncinola
Camanna
Boadilla Vorlf~lla convaJ/arla Epistylisplicati/is Zoothamnillm procmlU Opm:IIlarla c:oarcIala AspidiSCD cicada Trochilia ",iflllla ChilodoMlla uneinola Parameci_ aurelia Paramec/II", CQlldatu",
Cinelochllll", margarllaMI'" Deriotrlcha sp. LJtOltOlrU
Opm:II/arla artiClilaJa OpefCIIltria COt11'Cfa/a AspidiSCD cicada ChilodoMlla II1Icinola Deriostoma ClImpy/a TetmJrymena wt!omI/S Parameciu", caudotu", Uronema "igrlCtlfl.f
'-'/a
AciMrla uncinola
TrlI1l)'mlQ comprrSSllllf EncJte/yontor'pha vmn/C11Iarl.J Ak/opflS sp.
• Percentage considered higher than I%. Species listed in decreasing order ofabundance.
Biofilm characterization of several wastewater treatment plants
217
Different abundances of total ciliates were found along the RBC system in the 3 plants. In Las Matas and Boadilla. the second sampling point (corresponding to an intermediate stage of biological treatment) had the most populated biofilm (1.60 x 106 cil g-1 TS and 1.72 x 106 cil g'\ TS respectively). However, in Cam anna. the highest abundance of ciliates was at the fourth sampling point (last stage in the process) with an average number of 4.87 x 106 cil g-) TS. In this case, the great increase in abundance was due to the presence of Opercu/aria articulata and Aspidisca cicada in large numbers on just 4 sampling dates. oI,-r-----./
La, Mata'
.,/
80•
Penulchs
~f.iii'i~:;.cr Pentnchs
-"
60
Hl')lOIl"lOhs Cyrtophotlds
H)'!Il
40· 20 -
5cutJCOCIh... PI''''O.tMUds
Prostonl.ds Suetonans
.,/
/
~
Hvpotnclls
Cynophonds
-.G~~lv~ s~: C7''/ P1ewostomJds ,/ ProstMud. / . SuetonlJl.
oL._ -----..t/ Heteroo.oh.
H,1CrOtnchs
I 2 3 //
Camarma /
100/ 8060•
40· 20-
40'
Q -../. ./ SuetonlJl. o~ Heter_ebs I 2 3 4
Figure I. Abundance (%) and distribution of ciliates groups at different sampling points in the
RBe plants.
The distribution of groups and their relative abundances through the system are shown in Fig. I. Results indicated a variation in the abundance groups depending on the stage of biological treatment. Las Matas and Boadilla had similar distribution patterns. In both plants, peritrichs were the most representative ciliates in all stages, having the lowest abundance at the last sampling point. The most abundanct peritrich species were Vorticella convallaria (Las Matas) and Epistylis spp. (Boadilla). The abundance of these species decreased towards the last stage where other species were favoured (i.e. Zoothamnium spp.). Also in both cases. pleurostomids reached their highest abundance in the last stage (mainly the species Litonotus lamella) while hymenostomes were most abundant in the first stage. The main difference between Las Matas and Boadilla concerned scuticociliates, which behaved oppositely throughout the system. This was due to the occurrence of the species Cinetochilum margaritaceum that appeared with the highest abundance in the last stage of Las Matas while at Boadilla it was mostly present in the first stage. Heterotrichs were present in amounts higher than I % only in Las Matas. Camarma showed a different spatial distribution and abundance of groups compared to the other plants (Fig. I). Peritrichs (mainly Opercu/aria articulata) were the most abundant ciliates but only in the last stage of the process. Previous stages of treatment showed as hymenostomes dominant organisms at the beginning of the process and hypotrichs in the intermediate stages. Other groups well represented in the first contactor were scuticociliates (Uronema nigricans and Dexiotricha tranquilla), prostomids (Trimyema compressum) and suctorians (Enche/yomorpha vermicu/aris). The last two did not appear in significant amounts in the other plants. A gradual decrease of
218
B. PEREZ-UZ et al.
suctorians. scuticociliates, hymenostomes and prostomids was observed along the treatment stages in this plant. Good performance was found at Las Matas and Boadilla with an efficient BODs removal through the RBCs (55-70%). Both plants showed similar spatial distribution of ciliate communities. This could indicate that these communities might be associated with an efficient purification process. Nevertheless, Camarma showed lower diversity of ciliates with the dominance of only a few species. This may be related to poor performance, since the BODs removal by the biological process was only 20%. The occurrence of high numbers of certain species -Trimyema compressum and Enchelyomorpha vermicularis - which are especially adapted to poorly oxygenated conditions also suports this hypothesis. A previous study (Martfn-Cereceda et aI., 1996) in to activated sludge plants also located in Madrid (Spain), has allowed us to compare ciliate communities from both types of wastewater treatment plant. Peritrichs are the most representative ciliates in these processes. Species like Paramecium caudatum. P. aurelia, P. putrinum, Metopus sp., Brachonella spiralis, Trimyema compressum, Colpoda aspera and Enchelyomorpha vermicularis reported by other authors (Madoni and Ghetti, 1981; Augustin and Foissner, 1992) in activated sludge have only been found by us in RBCs. CONCLUSIONS RBC plants with high BODs removal efficiencies had richer populations of ciliate species than plants with poor treatment efficiency. Peritrichs made up the highest proportion of ciliates in these ecosystems. The careful characterization of ciliate communities inhabiting RBC biofilms performed in this research improves the knowledge of the role of these microorganisms in wastewater treatment plants. ACKNOWLEDGEMENTS This work was financially supported by C.A.M. (Project 1+0 0125/94). REFERENCES Augustin. H. and Foissner. W. (1992). Morphologie und Clkologie einiger Ciliaten (Protozoa: Ciliophora) aus dem Belebtschlamm. Arch. Protistenkd.• 141(4), 243-283. Chung. J. and Strom, P. (1991). Microbiological study of ten New Jersey rotating biological contactor wastewater treatment plants. Res. J. Wat. Pollut. Control. Fed.• 63(1),35-43. Curds. C. R. (1993). Interactions Protozoaires·Bacteries dans les methodes aerobies de lraitement des eaux usees. Ann. Bioi.• 32(1 ).1-12. Kinner. N. E. and Curds, C. R. (1987). Development of protozoan and metazoan communities in rotating biological contactor biolilms. Wat. Res., 21(4),481-490. Kinner, N., Curds. C. R. and Meeker, L. D. (1989). Protozoa and Metazoa as indicators of effluent quality in rotating biological contactors. Wat. Sci. Tech .• 20(11112).199-204. Fernandez·Galiano. D. (1994) The ammoniacal silver carbonate method as a general procedure in the study of protozoa from sewage (and other) waters. Wat. Res., 28(2). 495-496. Madoni, P. and Ghelli. P. F. (1981). The structure of ciliated protozoa communities in biological sewage·treatment plants. Hydrobiologia. 83, 207-21 S. Manln·Cereceda. M., Serrano, S. and Guinea. A. (1996). A comparative study of ciliated protozoa communities in activated· sludge plants. FEMS Microbiol. £Col.• 21. 267-276. Standard Methods for the Examination of Water Quality and Wastewater (1989). 17th edn, American Public Health Association/American Water Works AssociationlWater Environment Federation, Washinghton DC, USA. Wilbert. N. (1975). Eine verbesserte Technik der Protargolimpragnation fUr Ciliaten. Mikrokosmos, 64. 171-179.