1. Community Comparison of Nitrifying Microorganisms Between Aquaculture and Aquaponic Systems
Poster #296
Ryan P. Bartelme, Fred P. Binkowski, Sandra L. McLellan, Ryan J. Newton;
University of Wisconsin-Milwaukee, School of Freshwater Science, Milwaukee, WI
ABSTRACT: In recirculating aquaculture and aquaponic systems nitrification is critical to the removal of ammonia and nitrite, which are toxic to fish. These toxic nitrogen compounds are removed by cultivating ammonia and nitrite oxidizing microorganisms in a biofiltration component. Though ammonia and nitrite oxidizing microorganisms exhibit a mutualistic relationship, within these systems little is known about the primary nitrifying organisms and their relative abundance in the total bacterial community of a system. Here we sought to characterize the community of the University of Wisconsin - Milwaukee Recirculating Aquaculture System (UWM-RAS), which rears Perca flavescens, commonly known as Yellow Perch. The UWM-RAS was then compared to a second system, a commercial aquaponics venture (CAV), also contains Perca flavescens. High-throughput sequencing was used to profile the microbial community of both systems. The Illumina Hi-Seq V6 16s rRNA sequencing results indicated that Nitrosococcus spp. and Nitrosomonas spp. were the predominant ammonia oxidizing microorganisms (AOM) in the aquaponics biofilter. In the CAV, the main nitrite oxidizing microorganisms (NOM) were Nitrospira spp. UWM-RAS’s predominant AOM were Archaea, while the NOM were also Nitrospira spp. Both systems contained anaerobic ammonia oxidizing (ANAMMOX) bacterial species, which were detected in the solid waste clarifier in the UWM-RAS and in the biofilter of SWO’s system, but their contribution to the nitrogen budget of these systems remains unknown. With this research, we aim to more thoroughly understand the community composition and dynamics of nitrogen cycling microorganisms, so that system managers may more accurately derive a nitrogen budget for their particular system, regardless of which AOM or NOM are present.
INTRODUCTION:
METHODS:
RESULTS:
DISCUSSION:
UWM RAS bacterial community structure varies from component to component
UWM RAS exhibits a nitrifying community reflective of a soil environment
CAV bacterial community assemblage contains taxa commonly found in freshwater lakes
Management strategies and biosecurity measures may account for the differences in bacterial community structure
FUTURE EXPERIMENTS
Construct clone libraries for denitrification marker genes and nitrogen fixation genes
Find Carbon to Nitrogen Ratio and mass balance system components
Hi-throughput sequencing of Archaeal 16s rRNA
Determine ratio of Archaea to Bacteria via qPCR
Recirculating aquaculture and aquaponic systems are reliant upon microorganisms to facilitate the remediation of nitrogenous fish waste. In this study we sought to investigate the composition of the nitrifying microbial communities, to assess the relative abundance of Nitrosomonas spp. and Nitrobacter spp. in systems containing Perca flavescens, or yellow perch.
Nitrification rate equations for Nitrosomonas spp. and Nitrobacter spp. are often utilized in both aquaculture and aquaponic system design (1). However, it has been previously demonstrated that Archaea represent a portion of the microbial taxa that facilitate the nitrifying ammonia oxidation reaction in soil, and in recirculating aquaculture systems (2-4). Additionally, it has been shown Nitrospira spp., rather than Nitrobacter spp. are the dominant nitrite oxidizing taxa across multiple environments, including aquaculture systems (3, 5-6).
This study sampled the UWM Recirculating Aquaculture system (UWM-RAS), at the points indicated by red X’s in the UWM RAS System Diagram. However, it should be noted that the clarifier was only able to be sampled during the daily back flushing of the solid waste to the municipal sewer line. All other sites were sampled directly.
The commercial aquaponics venture (CAV) also raising Perca flavescens, was sampled at the rearing tank, biofilter, and solids clarifier. This study focuses on characterizing the nitrifying community in the UWM RAS, and compares the total community composition of the UWM RAS against a commercial aquaponic system.
Sample Sites
UWM RAS System Diagram
DNA Extraction – MP Bio Soil Kit
Illumina Hi-Seq V6 rRNA Sequencing
PCR Screen for Gene of Interest
Community Data Analysis
TOPO Cloning of Amplicons & Clone Library Construction
Clone Library Analysis
Dendrogram comparing the Relatedness of the UWM RAS’s System Component Bacterial Communities Using a Bray-Curtis Distance Matrix
Nitrospira nxrB clones added Parsimoniously nxrB ARB database (8)
Taxonomic Assignments by GAST
Archaeal amoA clones added Parsimoniously to Archaeal amoA ARB database (7)
REFERENCES
1. Timmons, Michael B.; Ebeling, J. M. Recirculating Aquaculture. (Ithaca Publishing Company, LLC, 2013).
2. Leininger, S. et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442, 806–9 (2006).
3. Brown, M. N., Briones, A., Diana, J. & Raskin, L. Ammonia-oxidizing archaea and nitrite-oxidizing nitrospiras in the biofilter of a shrimp recirculating aquaculture system. FEMS Microbiol. Ecol. 83, 17–25 (2013).
4. Morita, T., Andoh, T., Sakami, T. & Yamamoto, Y. Phylogenetic diversity of ammonia-oxidizing archaea and bacteria in biofilters of recirculating aquaculture systems. Mar. Genomics 7, 27–31 (2012).
5. Sakami, T., Andoh, T., Morita, T. & Yamamoto, Y. Phylogenetic diversity of ammonia-oxidizing archaea and bacteria in biofilters of recirculating aquaculture systems. Mar. Genomics 7, 27–31 (2012).
6. Kruse, M. et al. Relevance and diversity of Nitrospira populations in biofilters of brackish RAS. PLoS One 8, e64737 (2013).
7. Pester, M. et al. amoA-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions. Environ. Microbiol. 14, 525–39 (2012).
8. Pester, M. et al. NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira. Environ. Microbiol. (2013). doi: /
% Relative Abundance of Top 15 Bacterial Families Across Aquaculture and Aquaponic Systems Rearing Perca flavescens
CONTACT INFORMATION:
Ryan Bartelme,
UWM - School of Freshwater Science
600 E Greenfield Ave 53204
Acknowledgements:
Thanks go to Ryan Newton, Jen Fisher, and Sandra McLellan for their assistance learning bioinformatics, and to Fred Binkowski for access to the RAS System.
R bar chart generated with the help of Jen Fisher.
Funding was provided by the University of Wisconsin – Milwaukee School of Freshwater Science