Improvement of Acetobacter pasteurianus Fermentation Performance through Protoplast Fusion Technology
Native protoplast fusion technology was applied to obtain strains with high ethanol and acetic acid tolerance and acid production capability.The influence of compositions,enzyme hydrolysis time,enzyme hydrolysis concentration,and enzyme hydrolysis temperature on the preparation and regeneration of protoplasts were determined.Using protoplast fusion technology,the A.pasteurianus 1.41 with the high acetic acid-tolerance were fused with A.pasteurianus 503 with the high ethanol-tolerance.Then,the fusant were screened out,and ethanol tolerance,acetic acid production capability,and genetic stability were studied.The results indicate that the optimal lysozyme concentration,hydrolysis time and temperature were 50 μg/mL,40 minutes and 37 ℃,respectively.Using a high-osmotic solution composed of 10％sucrose,0.5 mol/L EDTA and 0.1 mol/L Tris-HCI,the protoplast preparation and regeneration rates were from 98.50％to 99.12％and from 40.51％to 42.70％,respectively.The fused strain RH-7 was demonstrated to grow at 13％ethanol or 1.5％acetic acid.In a fermentation containing 10％ethanol,RH-7 exhibited the highest acid production reaching 45.78 g/L.Genetic stability experiments revealed that strain RH-7 exhibited good stability in both growth and acid production.This study provides the optimal conditions for the preparation of A.pasteurianus protoplasts and identifies a strain RH-7 with excellent fermentation performance.The study contributed to an improved application of acetic acid bacteria in industrial.
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