This study was carried out into unexpected nitrogen removal in a fixed bed partial nitrification process, which is an essential pretreatment process for anaerobic ammonium oxidation (anammox). Two statistical methods, multiple regression analysis and the response surface method by spline (RSM-method), were applied to identify the main operational factors for this efficient one-step nitrogen removal using the anammox process. The RSM-method shows the requirements for higher removal rate using explanatory variables with high contribution values. It was found that nitrogen removal rates were obtained under high aeration rates and high flux of influent. It will be possible to attain a high volumetric removal rate of 1.5-2.9 kg-NH(4)-N/(m(3).d) in this reactor. New anammox bacteria (Asahi-BRW) were detected in this swim-bed reactor, with few detected in the sludge from this reactor. Ammonium oxidizing bacteria, including many types of Nitrosomonas europaea, were detected in free sludge from this reactor.