In order to obtain the excellent 1,3-propanediol (1,3-PDO) producer from wild-type Clostridium butyricum, adaptive evolution was carried out to select the strain for fast growth. The most significant change was that fermentation time decreased from 36 h to 20 h after adaptive evolution. Thus, it led to the corresponding volumetric productivity of 1,3-PDO increasing from 0.97 to 2.14 (g/LÁh −1 ) which increased by 114%. Adaptive evolution was also applied to butyric acid tolerant strain selected based on the fast-growing one through a simple equipment. 1,3-PDO concentration increased from 40.28 to 66.23 g/L through fed-batch fermentation by butyric acid tolerant strain compared with the fast-growing one. In addition, the endpoint strain was successfully and steadily used in the 50-L scale fermentation. Thus, adaptive evolution is an excellent strategy which can help us select the fast-growing strain and reduce the negative effect from substrate and metabolite inhibition. (a) the morphology of Clostridium butyricum in the initial stage of Gen 0 adaptive evolution process. (b) The morphology of Clostridium butyricum in the last stage of Gen 0 adaptive evolution process. (c) The effect of 5 g/L butyric acid and acetic acid to the growth of Gen 4 strain. (d) Different growing status of Gen 0, Gen 1, Gen 2, Gen 3, and Gen 4 strain in seed culture contained 70 g/L glycerol. The "XMU" were written on the back of the each bottles. [Color figure can be viewed at wileyonlinelibrary.com] Figure 5. Time course of Gen 7 strain fermentation on 50-L scale. (a, b) Represent the concentrations of glycerol, 1,3-PDO (g/L), acids and OD in batch and fed-batch fermentation process.[Color figure can be viewed at wileyonlinelibrary.com]

BACKGROUND: The immobilization of multiple oxidoreductases has attracted a lot of research interest for its industrial application in eco-friendly production. Here, a novel immobilization strategy was developed using manganese ion chelated linear polyethyleneimine as artificial hybrid scaffold that allowed self-assembly of bifunctional oxidoreductase (glycerol dehydrogenase-NADH oxidase) via multipoint coordination interaction.RESULT: The immobilization formed a highly ordered polymer-metal-enzyme complex structure shaped like a leaf vein with 200-nm-wide nanowires. Preliminary results showed that the immobilization could not only retain but substantially increased the biological activity of the immobilized enzyme by 8.2-fold. The equilibrium concentration of the reaction product DHA also was increased by 4.5-fold compared with that of the free enzyme, exhibiting higher elimination efficiency of product inhibition compared with other enzyme immobilization strategy using nanoparticles or activated agarose beads. At the same time, linear polyethyleneimine (LPEI)-manganese scaffolds could prevent enzyme subunit disassociation and stabilize the active multimeric form, which improved catalytic condition adaption and thermal stability of target enzyme. CONCLUSION: These results indicated a great potential of polymer-metal-enzyme hybrid in the enzyme immobilization and have provided a simple and efficient strategy for the immobilization of multiple oxidoreductases.