Microbial biofilm can be defined as multi-cellular aggregates adhering to carrier material,and has been widely used in water restoration.Despite prior research,the metabolic function of biofilm remains poorly understood.Using Eco-substrates as biofilm support,we evaluated the carbon requirements of microbial biofilm communities during biofilm formation(0,15,30,45,60 d)in the grass carp culture pond by Biolog-ECO plates.Our result showed that average well color development(AWCD)reached stationary phase after 168 h cultivation in all five selected sampling stages,and there were significant differences in sole carbon utilization characterized as AWCD value among five stages,and the higher carbon utilization capacity was observed in biofilm communities from 15 d to 45 d,it is higher than that observed in 0 and 60 d significantly.And the diversity indexes(Shannon index,Pielou index,McIntosh index,richness index)had the same rule of dynamic changes with AWCD.In addition,the polymers' and cabchydrates' utilization capacity of biofilm microbes was significantly higher than that of Phenolic compounds,carboxylic acids,amino acids,amines in all five sampling stages.With the biofilm formation,the utilization of α-D-glucose-1-phosphate,L-serine,N-acetyl-D-glucosamine,Tween 40,D-mannitol,etc.in biofilm microbes improved.The principal component analysis of microbial metabolic characteristics in biofilm communities showed that principal components 1(PC1)accounted for 33.9%,and principal components 2(PC2)accounted for 21.1% respectively.Based on samples' distance,most similar characters in carbon metabolism of microbial community were found among 15,30 and 45 d,but with significant differences in 0 and 60 d.Microbial biofilm communities had the strongest metabolic ability from 15 d to 45 d in the grass carp culture pond,and utilized carbon sources selectively.