Because colony formation is essential to seek bacterial functions by the direct observation of phenotype, the diversification of colony formation for culturable bacteria is a big challenge in the research field of Environmental Biotechnology. In this study, the biodiversity of cultivable bacteria (colony or liquid culture) was compared by using Luria-Bertani (LB) medium and waste sewage sludge (WSS) under different dilutions and temperatures. When WSS was used as a bacterial source, whereas the highest number of colonies was found at the concentration of WSS (5%), a particular concentration of LB (10%) or WSS (1%) as a growth medium showed the best number of the operational taxonomic units (OTUs) of colonies. The results of bacterial community structure indicated that there are 1, 8, and 12 bacterial genera found uniquely in the agar plates of LB, 10% LB, and 5% WSS. By contrast, when palm oil mill effluent sludge was used as a bacterial source, the effect of dilution was different with WSS. When comparing the biodiversity between colonies and liquid culture, a high OTU value was observed in the colonies on the plate. In addition, 30 degrees C showed the highest number of colonies in LB, 10% LB, and 5% WSS whereas the best OTUs were observed at 37 degrees C for LB and 10% LB, and at 25 degrees C for 5% WSS. This study demonstrates the diversification of cultivable bacteria through the number of OTUs in diluted LB medium and WSS, which is beneficial to isolate a unique bacterial strain. Key points center dot Impacts of diluted LB medium and WSS for colony formation were determined. center dot Difference of concentration of LB and WSS made different effects on colony formation. center dot Temperature change affected on diluted LB and WSS as media.