ORIGINAL ARTICLES   

Minerva Biotecnologica 2014 September;26(3):127-35

Copyright © 2014 EDIZIONI MINERVA MEDICA

lingua: Inglese

Ultraviolet mutagenesis breeding of protoplast of ammonia-oxidizing bacteria

Dong Y. ^{1, 2}, Zhang Y. ¹, Sun L. ^{1, 2}, Cao W. ^{1, 2}

¹ China University of Mining and Technology, Xuzhou, Jiaugsu, P.R. China; ² Xuzhou Institute of Technology, Xuzhou, Jiaugsu, P.R. China

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 α-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 80μg/mL, reaction time of ampicillin sodium was 8 hours, final concentration of lysozyme was 1 μg/mL, hydrolysis temperature of lysozyme was 40 °C, hydrolysis time of lysozyme was 30 minutes. Yield of protoplast was 4.35×105 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.