Ultraviolet mutagenesis breeding of protoplast of ammonia-oxidizing bacteria

Aim. Aim of the study was to obtain ammonia-oxidizing bacteria strain with strong capacity of ammoxidation. Methods. The strains were screened by repeated enrichment and separation methods from activated sludge. Physiological, biochemistrical and molecular biological methods were used to identified strains. Specific primers of gene amoA, encoding alpha-subunits of ammonia monooxygenase, were designed to amplify screened strains DNA. Results. Sequenced results of PCR products showed that 98% homology with Nitrosomonas sp. GH22 on Genbank. Ammonia-oxidizing bacteria were cultured at logarithmic growth phase, the sixth day. Final concentration of ampicillin sodium was 80 mu g/mL, reaction time of ampicillin sodium was 8 hours, final concentration of lysozyme was 1 mu g/mL, hydrolysis temperature of lysozyme was 40 C, hydrolysis time of lysozyme was 30 minutes. Yield of protoplast was 4.35x10(5) per ml and regeneration rate of protoplast was 0.0245% at above conditions. After cultivation of 250 mL, 500 mL and 1 L simulation sewage medium, the mutagenic strain UV003, radiated by ultraviolet 30 seconds, still had better capacity of ammoxidation with 85.2% removal rate of ammonia nitrogen. Conclusion. Physiological, biochemistrical and molecular biological identification synthesis results showed that the screened strain was Nitrosomonas sp. Ammonia-oxidizing bacteria strain with strong and stable capacity of ammoxidation was screened by ultraviolet mutagenesis breeding of protoplast.