Eutrophication in receiving waters due to the presence of nutrients including nitrogen is a well recognized environmental problem worldwide.Biological processes using a combination of nitrification and denitrification have long been utilized for nitrogen removal from wastewater.Because of the low growth rate and poor cell yield of nitrifying bacteria,including ammonia oxidizers (AOB) and nitrite oxidizers (NOB),nitrification is generally a rate-limiting step in biological nitrogen removal processes.Molecular biology techniques have allowed researchers to examine facets of natural microbial community that were previously inaccessible by traditional culturing methods.Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) has been demonstrated very useful for analyzing the microbial community structure of non-culturable environmental samples.Additionally,the treatment performance is likely to be strongly affected by the microbial composition,it is essential to link operational factors and N-removal performance with nitrifying bacteria community.In this study,the variations of nitrifying bacteria communities in A/A/O process with external manipulations were investigated by PCR-denaturing gradient gel electrophoresis.The external manipulations included temperature,loadings (F/M) and low C/N ratio.This aims of this research were ( 1 ) to ascertain the micro-ecological characteristics and establish the coupling relations between N-removal performance and key functional bacteria community in A/A/O process;(2) to study the effects of temperature,F/M,and C/N ratio on the nitrification performance and nitrifying bacteria community.The community structure of nitrifying bacteria varied notably with the fluctuations of temperature.The species of community were relatively abundant under temperature below 10℃,while the community structure was stable under temperature above 30℃.The optimal temperature was 25～30℃ for the effective nitrogen removal.When the F/M was increased stepwise from 0.03 to 0.44 g BOD5/g MLSS.d,the ammonia oxidizers and Nitrospira fluctuated hugely as measured by DGGE analysis.Not only the mere presence and absence of DNA bands were affected but also their relative abundance.However,the F/M had a relatively low influence on the Nitrobacter species.The optimized F/M ranged 0.14～0.28 g BOD5/g MLSS.d,and the Nitrobacter was identified as the predominant species in the A/A/O process.The variations of nitrifying bacteria under low C/N shocking and restoration were tested.The results showed the nitrification performance became worsening when the C/N ratio shocking was used,as evidenced by the disappearance of some ammonia oxidizers,nitrobacter and nitrospira.Some methods including the increase of reflux ratio and the shortening of sludge retention time were employed to restore the worsening system.The increase of reflux ratio ( the inner reflux ratio and the sludge reflux ratio were increased two-fold,respectively) could achieve the shortening of restoration time,and restore the nitrifying bacteria to the normal level.The contrast between pre-and post-restoration demonstrated the shortening of sludge retention time could effectively restore the ammonium oxidizers and nitrobacter species However,the nitrospira species could not be restored to the normal level.Therefore,the increase of reflux ratio was deemed as an effective restoration measure.Taken together,this research may be of great significance to provide valuable information for actual A/A/O wastewater processing systems.