Co-culture systems containing two microorganisms for production of useful substances are described. The co-culture of Bifidobacterium longum and Propionibacterium freudenreichii, where lactic acid produced once from lactose by B. longum is converted to acetic and propionic acids by P. freudenreichii, was carried out. Through the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic and propionic acids without lactic acid is produced. The antimicrobial activity of the mixture is higher than that obtained in the cultivation of B. longum alone. We developed a novel co-culture system composed of two fermenters and two microfiltration modules for efficient ethanol production from a mixture of glucose and xylose by co-culture of Pichia stipitis and Saccharomyces cerevisiae. The proposed co-culture system allowed regulation of the dissolved oxygen concentration at a level suitable for an individual yeast in each fermenter, as well as the successful exchange of culture medium between two fermenters. When P. stipitis and S. cerevisiae are cultivated individually under different oxygen supply conditions in the new co-culture system, the yield and productivity of ethanol from a glucose and xylose mixture are higher than in single culture of P. stipitis alone. By clarifying the interactions among microorganisms, new bioprocesses in which similar performance to co-culture systems is expressed even using a single microorganism, are expected to be developed for improvement of biochemical reaction systems.