Journal of Environmental Science and Health Part A (2007) 42, 517–523 Copyright C  Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520701189729 Identification of nitrifiers in a full-scale biological treatment system using fluorescent in situ hybridization OZGE EYICE, 1,2 BAHAR K. INCE, 3 GULNUR COSKUNER, 4 SEVAL SOZEN 1 and ORHAN INCE 1 1 Istanbul Technical University, Environmental Engineering Department, 34469, Maslak-Istanbul, Turkey 2 Istanbul Technical University, Molecular Biology and Genetics Department, 34469, Maslak-Istanbul, Turkey 3 Bogazici University, Institute of Environmental Sciences, 34342, Bebek-Istanbul, Turkey 4 Cumhuriyet University, Environmental Engineering Department, 58140, Sivas, Turkey Diversity of nitrifying bacterial population was investigated in sludge samples taken from a full-scale biological wastewater treatment plant (WWTP) treating domestic wastewater by fluorescent in situ hybridization (FISH) during seasonal operation. Duplicate grab samples were collected in March 2003, June 2003, December 2003 and May 2004 from the aerobic tank of the treatment plant. FISH results were interpreted with system performance in terms of BOD 5 , TKN and NO 3 -N removals and also with operational parameters such as wastewater temperature and sludge age. BOD 5 removal efficiencies were always greater than 90% whilst TKN removal in a range of 69–95% were achieved during the monitoring period. Although there were variations in operational conditions of the biological treatment system both Nitrosomonas and Nitrosospira genera from AOB and Nitrobacter genus from NOB were found to be present in all samples examined. Keywords: Biological wastewater treatment, fluorescent in situ hybridization, nitrification, ammonia-oxidizing bacteria, nitrite- oxidizing bacteria. Introduction Biological nitrification-denitrification is the most widely practised approach for nitrogen control in wastewater treat- ment. Since ammonia is toxic to aquatic organisms and the oxidation of ammonia in receiving water exerts a significant oxygen demand, nitrification process attracts a great deal of attention. [1] Nitrification, which is the first stage of nitro- gen removal, has two steps: oxidation of ammonia to nitrite and oxidation of nitrite to nitrate. These two steps are car- ried out by autotrophic nitrifying bacteria that have specific members to perform each step. As ammonia-oxidizing bac- teria, Nitrosococcus, Nitrosomonas, and Nitrosospira genera have been described based on the high similarity between the16S-rRNA genes. [2] All isolated chemolithoautotrophic, nitrite-oxidizing bacteria (NOB) belong to one of the four different genera Nitrobacter, Nitrococcus, Nitrospina, and Nitrospira. Performance of the wastewater treatment plants is gen- erally linked to the microbial community of the plant; Address correspondence to Bahar K. Ince, Institute of Environ- mental Sciences, Bogazici University, Istanbul, Turkey. E-mail: bahar.ince@boun.edu.tr Received April 13, 2006. therefore, it is important to study the microbiology of the activated sludge. However, it has long been realized that traditional culture-based methods such as most probable number [3] and selective plating [4] for determining the di- versity of microorganisms in environmental samples are unreliable. [5] These methods fail to be representative of the true natural diversity and are also time-consuming. [6] Often, selective enrichment cultures fail to match the conditions that many microorganisms require for growth in their nat- ural environment. [7,8] It is also known that there are many species which have not been isolated yet due to lack of suit- able methods. Since nitrifiers determine the sludge age of the biologi- cal treatment systems for nitrogen removal, it is very im- portant to identify the growth rate of these bacteria. The growth rate of nitrifiers mostly depends on different envi- ronmental factors such as, temperature, pH, and inhibitory substances which necessitates the characterization of the microorganism under each specific condition. The conven- tional methods above mentioned are not mostly used for the identification of nitrification bacteria, because of their long generation times to form detectable colonies and sensitivity to environmental factors. [9] In order to overcome the disadvantages of traditional culture-based methods, molecular tools such as denaturant