Electrochemically　active　biofilms　(EABs)　respire　in　a　unique　form　in　which　they　utilize　solid　external　materials　as　terminal　electron　acceptors　for　their　metabolism.　When　microorganisms　use　anodes　of　microbial　fuel　cells　(MFCs)　or　microbial　electrolysis　cells　(MECs)　and　so　on　for　respiration,　they　are　called　＇anode-respiring　biofilm　(ARB)＇.　There　were　complex　bio-electrochemical　and　mass　transfer　processes　as　ARBs　grew　in　bioreactors.　Currently,　very　little　real　time　data　is　available　characterizing　charge　and　mass　transfer　impedance　during　ARB　formation.　In　the　present　study,　ARB　was　cultivated　in　a　three-electrode　bioreactor　for　about　one　month.　We　measured　charge　and　mass　impedance　to　find　out　the　limiting　factors　for　power　output　through　different　phases　of　ARB　growth.　Electrochemical　impedance　spectroscopy　(EIS)　was　also　used　to　investigate　the　charge　and　mass　transfer　impedance,　while　Cyclic　Voltammetry　(CV)　was　used　to　measure　the　electrochemical　activities.　CV　and　EIS　were　all　measured　at　five　different　times　(80,　280,　430,　460　and　500　h)　during　ARB　formation.　Current　density　curve　shows　that　power　output　of　ABR　increased　as　biofilm　grew　and　it　goes　up　from　0.35　A　m-2　at　280　h　to　9.41　A　m-2　at　500　h.　CVs　also　illustrated　that　the　electron　transfer　rate　of　ABR　increased　greatly　over　time.　We　can　observe　that　the　limiting　current　density　from　CVs　has　a　similar　trend　with　the　current　curve　of　ARB　which　demonstrates　that　the　current　generation　ability　of　ABR　increases　during　biofilm　formation.　Impedance　value　of　anode-respiring　biofilm　displays　that　ohmic　resistance　is　around　25　Ω　showing　little　change.　However,　charge　transfer　impedance　goes　a　downward　trend.　They　are　12500,　2346,　996,　520　and　132　Ω　respectively　at　these　five　different　time　which　drops　sharply　with　biofilm　growing.　The　reason　is　that　the　microorganisms　inside　ARB　increases　significantly　especially　operating　after　460　h,　which　increases　the　substrate　utilization　rate　and　electron　transfer　rate　of　ABR　greatly.　However,　mass　transfer　impedance　shows　a　upward　trend　over　time.　We　found　that　charge　transfer　resistance　accounts　for　99.8%　and　72.9%　respectively,　at　80　and　460　h.　But　it　keeps　dropping　to　just　around　33.1%　at　500　h,　which　demonstrates　that　charge　transfer　process　is　the　limiting　factor　of　current　production　at　the　beginning　of　operation.　But　it　keeps　dropping　to　just　around　33.1%　at　500　h　proving　that　mass　transfer　process　gradually　plays　an　important　role　in　power　output　of　ARB　when　biofilm　is　mature.　Therefore　different　methods　to　be　used　for　reducing　the　charge　or　mass　transfer　resistance　during　ARB　formation　in　bio-electrochemical　system　is　of　great　importance　to　improve　its　electrochemical　performance.　©　2016,　Science　Press.　All　right　reserved.