heterotroph isolation presentaion
TRANSCRIPT
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