Development and application of a novel screening method and experimental use of the mutant bacterial strain Clostridium beijerinckii NCIMB 8052 for production of butanol via fermentation of fresh cassava

Abstract

Classic chemical mutagenesis has a demonstrated potential to create a strain of Clostridium with improved fermentation performance for obtaining high butanol yield. However, screening methods to select the desired bacterial phenotype from large populations are not currently feasible or readily available. In this paper, a simple, novel screening method to select target strains by using trypan blue dye as an indicator in solid starch media to detect the target mutant bacterium was devised. A well performing mutant strain Clostridium beijerinckii no. 15 was obtained using this screening method. It was derived from wild-type C. beijerinckii NCIMB 8052 by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenesis, and its fermentative capability for producing butanol was investigated, using hydrolysate of fresh cassava and other substrates. The mutant strain C. beijerinckii no. 15 produced a maximum yield of 15.06 g L⁻¹ butanol and 23.78 g L⁻¹ total solvents, when the fresh cassava substrate was given a simple pretreatment at 121 °C for 1 h. For other substrates, the highest butanol yields: 20.67 g L⁻¹ from glucose, 17.96 g L⁻¹ from liquefied dextrin, and 13.59 g L⁻¹ from liquefied corn powder (P ≤ 0.05) were also achieved. Experimental trials showed that the mutant strain had a significantly greater fermentation ability, and produced a high-yield butanol from fresh cassava hydrolysates. Further research showed that the highest overall yield of butanol resulted from the improvement of enzymatic activity of C. beijerinckii no. 15's α-amylase (P ≤ 0.05). Also, the protein structure of the α-amylase was predicted and its amino acid mutation sites were analyzed. Results revealed that these mutant amino acids may have somehow improved α-amylase activity and thus, led to producing more butanol. Our research will contribute to efforts to develop C. beijerinckii fermentation of fresh cassava to produce butanol.