PK-192 (JTK):

Molecular approach for detection of anaerobic anmmonium-oxidizing bacteria in sludge for livestock wastewater

The purpose of this study was to identify anaerobic ammonium-oxidizing bacteria and to support quantitative PCR results which were performed for searching an effective seeding sludge to achieve successful enrichment of anaerobic ammonium-oxidizing bacteria. Eleven sludge-samples originated from ten purification facilities for livestock wastewater were used in this study. At first, six samples among the eleven samples were selected by gas productivity per a gram biomass. The selected samples were used for the experiments of qPCR and 454 pyrosequencing after extraction of the genomic DNAs. The quantitative PCR was done with the primer sets of AMX818F/AMX1066R and HZO2aF/HZO2aR. The amplicons for pyrosequencing analysis were constructed by using the primer-set which is consisted of a specific pla46F primer targeting the conserved 16S rRNA gene belongs to Plantomycetes phylum and eub 518R targeting the conserved 16S rRNA genes of all bacteria. There is no meaningful correlation between the results of qPCR using both PCR primer sets, which indicate that the coverage of the primer sets would be different from anaerobic ammonium-oxidizing bacteria. According to the alignment of pyrosequencing results, all samples showed the plantomycetes-dominant bacterial community structure indicating the used primer-specificity. The phyla of “Chloroflexi” and “Lentisphaerae” were followed as the substantial groups in the order by the usage of the primer-set. However, most of the amplicons classified as plantomycetes were classified as uncultured environmental samples on the genus level without showing any information about the closest well-known anaerobic ammonium-oxidizing bacterial group in this study.
This project is supported by Korea Ministry of Environment as “program for promoting commercialization of promising environmental technologies”

keywords:anaerobic ammonium oxidizing bacteria,plantomycetes,pyrosequencing,qPCR