Appl Microbiol Biotechnol (2006) 70: 47–52 DOI 10.1007/s00253-005-0046-2 BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Kazuichi Isaka . Yasuhiro Date . Tatsuo Sumino . Sachiko Yoshie . Satoshi Tsuneda Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor Received: 3 April 2005 / Revised: 8 May 2005 / Accepted: 30 May 2005 / Published online: 14 July 2005 # Springer-Verlag 2005 Abstract The doubling time of anaerobic ammonium-oxi- dizing (anammox) bacteria in an anaerobic biological fil- trated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count an- ammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1×10 6 and 1.7×10 7 cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (μ) was 0.39 day -1 . This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly dem- onstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria. Introduction The anaerobic ammonium-oxidizing (anammox) reaction is a novel biological denitrification that utilizes nitrite and ammonium at the same time (Mulder et al. 1995; Van de Graff et al. 1997; Strous et al. 1997, 1999a). The stoi- chiometry of the anammox process obtained from experi- mental data is as follows (Strous et al. 1998): 1NH þ 4 þ 1:32NO  2 þ 0:066HCO  3 þ 0:13H þ ! 1:02N 2 þ 0:26NO  3 þ 0:066CH 2 O 0:5 N 0:15 þ 2:03H 2 O Anammox reaction, in which ammonium is converted to nitrogen gas as a hydrogen donor, does not require any addition of a hydrogen donor such as methanol. In addition, the anammox process achieves very high nitrogen removal rates up to 8.9 kg N/m 3 per day (Sliekers et al. 2003), which could make the reactor capacity smaller than that of con- ventional systems. From these advantages, the anammox process was considered as an economical and effective process. It was reported that members of Planctomycetales are predominant in anammox sludge capable of the anammox reaction (Strous et al. 1999b), and the growth rate of an- ammox bacteria was extremely low. To apply the anammox reaction to actual nitrogen removal in wastewater treatment process, a large amount of seed sludge is necessary. How- ever, too slow a growth becomes a problem in practical use. At first, the doubling time of anammox bacteria was re- ported as 30 days (Van de Graff et al. 1996), but after that, it was corrected to 11 days (Strous et al. 1998). On the other hand, in the water treatment test using anammox bacteria, we have sometimes observed that the nitrogen removal rate increases by more than twofold during 11 days (data not shown). Then, the question has been raised as to whether or not the reported doubling time of anammox bacteria is true. In previous reports (Strous et al. 1998; Van de Graff et al. 1996), the growth rate of anammox bacteria was calculated from biomass yield and nitrogen removal rate. The in- creasing rate of nitrogen load during cultivation was so low that doubling time of anammox bacteria was as low as that in their stationary phase. Further, there is no data for the K. Isaka (*) . T. Sumino Hitachi Plant Engineering and Construction Co., Ltd., Kami-Hongo 537, Matsudo, Chiba, 271-0064, Japan e-mail: k-isaka@hitachiplant.co.jp Tel.: +81-47-3616103 Fax: +81-47-3616107 Y. Date . S. Yoshie . S. Tsuneda Department of Chemical Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo, 169-8555, Japan