After a test trial of four years at the Free University of Barcelona (Spain), the MELiSSA CIII packed-bed pilot plant proved to be a functionally stable nitrifying bioreactor by efficiently converting high loads of ammonia to nitrate. The packed-bed bioreactor was dismantled and biomass stored as lyophilized and frozen samples originating from eight positions along the vertical feed-axis of the bioreactor. The nitrifying activity of the reactivated samples was tested using a colorimetric indophenol ammonia analysis; the bacterial community was analyzed using a PCR-based Denaturing Gradient Gel Electrophoresis (DGGE) approach, targeting both 16S rRNA- and ammonia monooxygenase (amoA)-genes in lyophilized and frozen samples, reactivated lyophilized samples, and on cDNA originating from reactivated frozen samples. The presence of mobilizing plasmids was screened using a PCR-based IncP-specific approach and a qualitative exogenous mobilizing plasmid isolation approach, based on replica mating. The indophenol ammonia analysis demonstrated the absence of nitrifying activity in seven of the eight inoculated lyophilized samples, demonstrating a sensitivity of ammonia-oxidizing bacteria due to the lyophilization process. The frozen samples showed normal nitrifying activity. DGGE analysis on lyophilized samples demonstrated the CIII bioreactor community to be enriched in diversity, and yielded complex community profiles. Eight isolates were obtained from the nitrifying lyophilized sample and six were identified. Both gram-positive as gram-negatives were detected in the bioreactor, among which some might have a stabilizing effect on the functionally stable reactor. Approximately six organisms were observed to be active in ammonia-rich and nitrite-rich media. Surprisingly, no N. europaea was detected on cDNA of ammonia-enriched medium cultures, despite the observed nitrifying activity in the ammonia analysis. The PCR-based screening for IncP-plasmids demonstrated that nearly no IncP-plasmids were present, and no traG-harbouring plasmids were observed. No transconjugants could be detected with the qualitative triparental exogenous plasmid isolation, and as of yet no mobilizing plasmids could be detected in the bioreactor. The results indicate that the CIII nitrifying packed-bed pilot plant was a functionally stable ammonia-converting reactor, with high bacterial diversity and nearly devoid of mobilizing-plasmid-harbouring organisms.
|Place of Publication||Brussel|
|State||Published - 16 Sep 2005|