Construction of engineering bacteria degrading residual polyacrylamide in coal slime water by protoplast fusion technique

Polyacrylamide (PAM) is widely used as flocculant in coal preparation. A protoplast fusant with enhanced degradation ability of PAM was obtained by intergeneric protoplast fusion between Rhodopseudomonas spheroids and Bacillus subtilis. The protoplast preparation was optimised and inactivation of both parents was performed in different ways. The degradation abilities of domesticated R. spheroides, B. subtilis and mixed bacteria (v/v, 1.00) were compared with protoplast fusant. The results showed that the optimal dosage for R. spheroides protoplast preparation was determined to be 20.00% sucrose, 1.00 mg/mL lysozyme, 0.20% EDTA and 60.00 min. Under such conditions, the protoplast formation rate and the protoplast regeneration rate were 63.70% and 8.90%, respectively. The optimal dosage for B. subtilis protoplast preparation was determined to be 20.00% sucrose, 1.50 mg/mL lysozyme and 60.00 min. Under such conditions, the protoplast formation rate and the protoplast regeneration rate were 58.50% and 9.45%, respectively. The protoplast inactivation of R. spheroides and B. subtilis were optimally conducted by ultraviolet radiation (70.00 s) and by heat at 55.00°C (50.00 min), respectively. The degradation ability of protoplast fusant was obviously improved. Furthermore, using microscopic observation, it was found that both parents form protoplasts by the way of apical release.