Appl Microbiol Biotechnol (2006) 72: 182–189 DOI 10.1007/s00253-005-0235-z ENVIRONMENTAL BIOTECHNOLOGY Sachiko Yoshie . Hiroshi Makino . Hidenobu Hirosawa . Kosuke Shirotani . Satoshi Tsuneda . Akira Hirata Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater Received: 4 September 2005 / Revised: 22 October 2005 / Accepted: 26 October 2005 / Published online: 21 December 2005 # Springer-Verlag 2005 Abstract A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal- restriction fragment length polymorphism (T-RFLP) anal- ysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity condi- tions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitri- fication system for saline wastewater can be designed using acclimated sludge with a halophilic community. Introduction Halophilic denitrifying bacteria are quite useful in nitrogen removal from saline industrial wastewaters. Although the existence and function of several halophilic denitrifying bacteria have been reported so far (Caton et al. 2004; Peyton et al. 2001), these bacteria have been rarely utilized in the practical denitrification process for saline industrial waste- water. Currently, saline wastewater is diluted to low salinity to prevent the inactivation of bacteria in activated sludge and biofilms (Hirata et al. 2001; Diner and Kargi 1999). However, there are some problems with the practical use of these systems: poor denitrification performance for practical use, high operational cost, and augmentation of wastewater by the use of tap water for dilution. In our investigations to develop a saline industrial wastewater treatment system, an improvement in denitri- fication efficiency was observed at a high salinity in comparison with that at low salinity using long-acclimated sludge (Yoshie et al. in press). In our previous study, a microbial community in a conventional denitrification sys- tem (salinity: 1–3% of NaCl) was analyzed by PCR- denaturing gradient gel electrophoresis (DGGE; Yoshie et al. 2001) and fluorescence in situ hybridization (FISH; Yoshie et al. 2004a). Results indicated that bacterial species of the genera Halomonas, Marinobacter, and Pseudomo- nas, all of which belong to the γ-Proteobacteria, played some roles in denitrification under such low-salinity con- dition (1–3%). However, it is unclear how these pop- ulations change with an increase in salinity, particularly in a reactor with a high denitrification performance. Further- more, the optimal salinity range for halophilic species with a high denitrification activity should be determined for the design of a high-performance denitrification system. In this study, the salinity range within which denitrifica- tion efficiency could increase was determined by continuous treatment tests of metal refinery wastewater representative of saline industrial wastewater. Then, terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor the microbial community shift during acclima- tion to 10% salinity condition and to determine dominant S. Yoshie . H. Makino . H. Hirosawa . K. Shirotani . S. Tsuneda (*) . A. Hirata Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan e-mail: stsuneda@waseda.jp Tel.: +81-3-52863210 Fax: +81-3-32093680