BACKGROUND Physiological parameters (culture pH, aeration rate, and agitation speed) were optimized to improve gamma-PGA production by batch culture of Bacillus subtilis RKY3. The optimum conditions for recovery of gamma-PGA from highly viscous culture broth were also investigated. RESULTS Enhanced gamma-PGA production (41.4 g L-1, 397 cP) was observed under optimized physiological culture parameters (no pH control, 1.5 L min(-1) aeration, and 300 rpm agitation). The cells were completely removed by diluting the viscous culture broth 7.5-, 5.0-, and 2.5-fold at pH 6.0, 4.0, and 2.0, respectively. Maximum recovery of gamma-PGA was observed when the volume ratio of cell-free culture broth to cold ethanol was 1:5 at pH 6.0. gamma-PGA with a molecular weight kDa was successfully excluded in an ultrafiltration system to develop more economical recovery of gamma-PGA from cell-free culture broth, which resulted in efficient gamma-PGA concentration with a molecular weight >100 kDa. CONCLUSION The physiological parameters affecting gamma-PGA production were optimized, and the isolation and recovery of gamma-PGA from viscous culture broth was also evaluated. Cells were removed completely by diluting the culture broth, and precipitation of gamma-PGA using cold ethanol was better than that of other alcohols. Ultrafiltration was found to be an efficient recovery method for concentration of culture broth containing gamma-PGA. (c) 2013 Society of Chemical Industry