Identification of Heterotrophic Contaminants from Nitrifying bioreactors

Aidan Maxwell | Luis Sayavedra-soto Lab |May 2015

Outline

○ Why heterotrophs?

○ Seven heterotroph contaminants from three separate bioreactors:

○ N. europaea & N. winogradsky Consortium bioreactor

○ 2x N. hamburgensis bioreactors

○ Basic heterotroph characteristics

○ conclusion

Heterotrophic relationships

○ What are some characteristics of heterotrophs growing with nitrifying bacteria?

○ Denitrifying activity

○ Consumers of alternative products

○ Saprophytic or predatory bacteria

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

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

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

Sequencing Results: N. Hamburgensis contaminant

○ Gordonia terrae – 99%

○ Nitrilase – CN functional group hydrolysis

○ β-glucosidase – polysaccharide degradation

○ Benzothiophene desulfurization

Sequencing Results: N. Hamburgensis contaminant

○ Microbacterium oxydans – 100%

○ Diverse metabolism

○ Chitinase

○ Heavy metal water treatment

Sequencing Results: N. Hamburgensis contaminant

○ Bacillus horikoshii – 99%

○ Denitrification activity

○ Alkaliphilic

Sequencing results: Consortium contaminant

○ Shinella zoogloeoides – 99%

○ N2O reducer

○ Pyridine is sole C and N source

Sequencing results: Consortium contaminant

○ Flavobacterium lindanitolerans – 100%

○ No presence of nitrate reductase

○ Diverse metabolism

Sequencing results: Consortium contaminant

○ Achromobacter mucicolens – 99%

Sequencing results: Consortium contaminant

○ Stenotrophomonas maltophilia – 99%

○ Predatory

○ Diverse metabolism

○ Ubiquitous in aqueous environments

Conclusion and Future Studies

○ Heterotrophs are diverse

○ Sequence entire 16S rDNA?

○ Additional consortium experiments

Acknowledgements

○ OSU CGRB○ Luis Sayavedra-Soto Lab

○ Taylor Barns○ Brett Mellbye