Seven heterotrophic contaminants were isolated from three nitrifying bioreactors. The contaminants were identified via PCR amplification and sequencing of the 16S ribosomal subunit. From a Nitrobacter hamburgensis bioreactor that crashed, two contaminants were isolated and identified as Gordonia terrae and Microbacterium oxydans. From a stable N. hamburgensis bioreactor, one contaminant was isolated and identified as Bacillus horikoshii. From a Nitrosomonas europaea and Nitrobacter winogradskyi consortium bioreactor, four contaminants were isolated - Shinella zoogloeoides, Flavobacterium lindanitol

1. Identification of Heterotrophic
Contaminants from Nitrifying
bioreactors
Aidan Maxwell | Luis Sayavedra-soto Lab |
May 2015

2. Outline
○ Why heterotrophs?
○ Seven heterotroph contaminants from three separate bioreactors:
○ N. europaea & N. winogradsky Consortium bioreactor
○ 2x N. hamburgensis bioreactors
○ Basic heterotroph characteristics
○ conclusion

3. Heterotrophic relationships
○ What are some characteristics of heterotrophs growing with nitrifying bacteria?
○ Denitrifying activity
○ Consumers of alternative products
○ Saprophytic or predatory bacteria

4. N. hamburgensis Chemostat Experiments
○ N. Hamburgensis bioreactors:
○ 60 mM NaNO2 media
○ Nitrobacter hamburgensis: Nitrite oxidizer – Nitrate producer
○ One Crashed
○ Neutral effect
○ Three contaminants isolated
○ Two from the bioreactor that crashed
○ One from the bioreactor that had a neutral effect

5. N. europaea & N. winogradskyi consortium
experiment
○ Consortium Bioreactor:
○ 60 mM NH4+ media
○ Nitrosomonas europaea: Ammonium oxidizer – Nitrite producer
○ Nitrobacter winogradskyi: Nitrite oxidizer – Nitrate producer
○ Heterotroph contaminants present
○ Observed increase in OD600 of N. europaea and N. winogradskyi with contaminants
○ Four contaminants isolated
○ Two small colony & two large colony

6. Process of Identification
○ Contaminants were streaked onto LB agar plates directly from bioreactor aqueous media
○ 60% glycerol stocks of contaminants were made when individual colonies could be isolated on
plates. Stocks kept at -80˚C
○ DNA stocks made for all contaminants besides the two large colony contaminants
○ PCR for 16S ribosomal subunit sequence
○ Primers: BAC 311 & BAC 797 (486bp)
○ Sanger sequencing – OSU CGRB
○ BLAST the sequencing results

7. Sequencing Results: N. Hamburgensis
contaminant
○ Gordonia terrae – 99%
○ Nitrilase – CN functional group
hydrolysis
○ β-glucosidase – polysaccharide
degradation
○ Benzothiophene desulfurization

8. Sequencing Results: N. Hamburgensis
contaminant
○ Microbacterium oxydans – 100%
○ Diverse metabolism
○ Chitinase
○ Heavy metal water treatment

9. Sequencing Results: N. Hamburgensis
contaminant
○ Bacillus horikoshii – 99%
○ Denitrification activity
○ Alkaliphilic

10. Sequencing results: Consortium
contaminant
○ Shinella zoogloeoides – 99%
○ N2O reducer
○ Pyridine is sole C and N source

11. Sequencing results: Consortium
contaminant
○ Flavobacterium lindanitolerans – 100%
○ No presence of nitrate reductase
○ Diverse metabolism

12. Sequencing results: Consortium
contaminant
○ Achromobacter mucicolens – 99%

13. Sequencing results: Consortium
contaminant
○ Stenotrophomonas maltophilia – 99%
○ Predatory
○ Diverse metabolism
○ Ubiquitous in aqueous environments

14. Conclusion and Future Studies
○ Heterotrophs are diverse
○ Sequence entire 16S rDNA?
○ Additional consortium experiments

15. Acknowledgements
○ OSU CGRB
○ Luis Sayavedra-Soto Lab
○ Taylor Barns
○ Brett Mellbye