Some acetogenic bacteria has the ability to use carbon monoxide (CO) using the Wood-Ljungdahl pathway. Biological conversion of CO using acetogens offers benefits of utilizing industrial waste gases containing CO and generating valuable fuels such as ethanol, butanol and 2,3-butanediol. In this study, we screened 2,3-butanediol-producing microbes among various environmental samples and isolated a new strain, Clostridium sp. AWRP from a wetland. This strain is able to produce acetate, ethanol and 2,3-butanediol as major metabolites using CO. To improve 2,3-butanediol production, the wild-type strain was modified by random mutagenesis technology. After treatment of cells with N-methyl-N-nitro-N-nitrosoguanidine (NTG), mutants with better growth than the wild-type strain under high CO partial pressure were selected. They showed different growth and metabolite production patterns. Among them, a few mutants were identified to have improved CO consumption and 2,3-butanediol production. Here, we describe effective strategies for selection of CO-utilizing 2,3-butanediol producers through enrichment processes during isolation and mutagenesis.ls such as ethanol, butanol and 2,3-butanediol. In this study, we screened 2,3-butanediol-producing microbes among various environmental samples and isolated a new strain, Clostridium sp. AWRP from a wetland. This strain is able to produce acetate, ethanol and 2,3-butanediol as major metabolites using CO. To improve 2,3-butanediol production, the wild-type strain was modified by random mutagenesis technology. After treatment of cells with N-methyl-N-nitro-N-nitrosoguanidine (NTG), mutants with better growth than the wild-type strain under high CO partial pressure were selected. They showed different growth and metabolite production patterns. Among them, a few mutants were identified to have improved CO consumption and 2,3-butanediol production. Here, we describe effective strategies for selection of CO-utilizing 2,3-butanediol producers through enrichment processes during isolation and mutagenesis.1