Application of temperature gradient gel electrophoresis to the characterization of a nitrifying bioaugmentation...

The microbial population of a nitrifying bioaugmentation product (NBP) has been examined using a combination of conventional bacteriological methods and modern molecular techniques. Variable region 3 (V3) of the 16S rRNA genes of the bacteria in NBP was amplified via the polymerase chain reaction (PCR) with universal eubacterial primers and analyzed via temperature gradient gel electrophoresis (TGGE). Two of the predominant PCR products in NBP were purified from the TGGE gel matrix, reamplified via PCR and sequenced. Two nitrifying strains (NS500-9 and MPN2) that had been isolated from the NBP mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR-amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the NBP, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter species, respectively, in the database. Samples from a laboratory-scale bioreactor, bioaugmented with NBP, were used in an attempt to correlate an increase in activity with a detectable shift in the population of NS500-9 and MPN2 via TGGE. No detectable shift in population was observed in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when NBP was present in activated sludge. In biomass spiking experiments as well as in genomic DNA spiking experiments, it was found that NBP must be present at a level of at least 5% of the total population in order to be detected, whereas bioaugmentation at 1% of the total population was enough to yield significant improvements in nitrification efficiency. This study demonstrates how community profiling of an undefined microbial population via TGGE can be used to examine the biological importance of organisms isolated from the same mixed population.