Biofilm-based fermentation, as a new immobilisation strategy, is beneficial for industrial fermentation due to its excellent environmental resistance, high productivity and continuous fermentation relative to calcium alginate-immobilised fermentation. These two techniques differ mainly regarding cell stages. Here, we describe the cell phenotype ofSaccharomyces cerevisiaebiofilm-based fermentation and compare cell cycle stages with those during immobilisation in calcium alginate. Most cells in the biofilm-based fermentation adhered to the cotton-fibre carrier of the biofilm and were in the G2/M phase whereas alginate-embedded cells were in the G1/G0 phase. Deletion of theRIM15gene, which regulates cell cycle progression according to nutritional status, hampered the cell cycle arrest observed in alginate-embedded cells, enhanced biofilm formation and improved fermentation ability. The improved biofilm formation shown by therim15o strain could be attributed to an increase in the expression level of the adhesion proteinFLO11and synthesis of trehalose. These findings suggest that the extracellular environment is mainly responsible for the difference between biofilm-based fermentation and alginate-embedded fermentation, and thatRIM15plays an essential role in cell cycle progression.