1. Corianne Tatariw, Elise Chapman, Dr. Jennifer Edmonds
Exploring Community Dynamics of Denitrifying Bacteria in the Cahaba River
Corianne Tatariw, Elise Chapman,
Dr. Jennifer Edmonds
Department of Biological Sciences
University of Alabama, Tuscaloosa

2. Effects of N-loading Harmful algal blooms
Acidification/ eutrophication of freshwater systems
Loss of biodiversity
Harmful algal blooms
Eutrophication/ hypoxia in coastal systems
Images from NOAA

3. Goal: evaluate denitrification as a meaningful nitrate sink in large rivers
How does denitrifier community structure respond to environmental parameters?
What is the effect of these changes in denitrifier community structure on rates of denitrification?

4. Ecosystem Function and Diversity
High diversity of microorganisms suggests functional redundancy, but…
Denitrifier communities respond differently to environmental conditions, affecting rates of denitrification (Cavigelli and Robertson 2000)

5. NO3- NO2- NO N2O N2 gas Heterotrophic facultative anaerobes
nirS /nirK
gas
Heterotrophic facultative anaerobes
Dissolved Oxygen (DO) affects distribution of nirS and nirK denitrifiers (Graham et al 2010, Knapp et al 2009)
nirK denitrifiers can function under fluctuating DO conditions
nirS denitrifiers found in consistently anoxic environments

6. Sediment Particle Size
Measured as porosity: Fraction of void space in a volume of sediment
Greater surface area: volume in smaller sediments
Affects movement of DO through sediment

7. The Cahaba River VR FL CP Valley Ridge (VR) Coastal Plain (CP)
Fall Line (FL)

8. Porosity in the Cahaba

9. The Cahaba River: A Nitrate Sink
Birmingham
The Cahaba River: A Nitrate Sink

10. How does denitrifier community composition change over space and time?
NirS and nirK denitrifiers will have distinct community structures within each geophysical province due to changes in sediment size and channel slope.
Dominance of nirS vs. nirK will vary due to spatial and seasonal variations in DO

11. Methods Three subsamples taken from homogenized sediment in field
Spring, Early Summer, Late Summer, Winter
2 locations per geophysical province (VR, FL, CP)
TRFLP of nirS and nirK
nirS: cd3aF/R3cd-HEX
nirK: FlaCu-FAM/R3Cu
Restriction enzyme: MspI
Analysis of Similarity: Primer v.6

12. ANOSIM: nirS No significant difference by geophysical province or date
2D Stress = 0.14
No significant difference by geophysical province or date
nirS dominates in anoxic environments which may have less variable conditions
Valley Ridge
Fall Line
Coastal Plain
Global R = 0.157
p = 0.03

13. ANOSIM: nirK Coastal Plain different from Valley Ridge and Fall Line
No significant seasonal difference
CP has finer, sandy substrate compared to VR and FL
2D Stress = 0.22
Valley Ridge
Fall Line
Coastal Plain
Global R = p = 0.001

14. How do rates of denitrification vary over time and space in the Cahaba River?
Denitrification rates will vary among geophysical provinces in response to changes in denitrifier community composition.
Highest rates of denitrification will coincide with high primary productivity as carbon limitation is alleviated

15. Methods Sediment samples collected with microbial samples
Acetylene block method to measure denitrification potential rates

16. Denitrification Potential Ratesb ab ab
Denitrification Rate (mmol N2 g-1 hr-1) a

17. Implications The Cahaba River is a sink for NO3-
Lack of relationship between changes in community composition and denitrification rates suggests functional redundancy

18. Acknowledgements Committee Members Dr. Jennifer Edmonds
Dr. Robert Findlay
Dr. Lisa Davis
Lab Members
Elise Chapman
Courtney Dragiff
Susan Jozefiak
Ben Wilson
Field and Lab Help
Chau Tran
Julie Jarnigan
Diane Schneider
Marie Wilson

23. ANOSIM:16S rDNA at Fall Line 1
2D Stress = 0.16
Bacterial community structure changes over time through macrophyte growing season
2 June 2010
14 July 2010
24 June 2010
Global R = 0.414
p = 0.001

24. Nitrogen and Carbon Response: Winter/Spring*
Dec. 2010
DNP (nmol N2 g-1 hr-1)
Apr. 2011
DNP (nmol N2 g-1 hr-1)
* Statistically significantly different from ambient denitrification potential rate; p=0.05

25. The Cahaba River: An N Sink?
Fall Line

26. Humans and Nitrogen
Estimated deposition of reactive nitrogen in a) 1890s and b) 1990s
Fertilizer
Leguminous crops
Livestock
Fossil fuel combustion
Galloway et al Biogeochemistry 70:

27. Microbially Mediated N-removal
Burgin and Hamilton Front Ecol. Environ. 5: 89-96

28. Nitrite Reductase Functional Genes
nirS: Consistently anoxic environments, always active
nirK: Areas of DO flux, more active at night
Nitrite reductase cytochrome-cd1
Nurizzo et al Biochemistry 37:
Copper-containing nitrite reductase
Nojiri et al PNAS. 104:

29. Summary of DNP Data
Denitrification is occurring along the Cahaba at rates comparable to those in literature (Piña-Ochoa and Álvarez-Cobelas 2006)
Highest rates occurred at the Fall Line in summer
Lack of C response suggests in-stream primary producers alleviated C-limitation
Spatial heterogeneity at Fall Line

30. C and N Limitation – June 2010
Denitrification Potential (nmol N2 g-1 hr-1)

31. C and N Limitation – Sept 2010* * * * * * *
Significantly different from ambient DNP rate; p <0.05

32. Future Work
1) Denitrification potential rate measurements in April 2011 will be highest at the Fall Line
1) How do rates of denitrification vary in the Cahaba River?
2) How does denitrifier community composition change over space and time?
2) T-RFLP of nirS and nirK functional genes will show distinct denitrifier community structure at the scale of geophysical province.
3) qPCR of nirS and nirK will show increased denitrifier activity and abundance associated with higher denitrification rates and there will be changes in nirS vs. nirK activity over space and time.
3) How does denitrifier activity and abundance change over space and time?

34. Large and unregulated
North of Birmingham to Selma, AL
Confluence with Alabama River
Valley Ridge, Fall Line, Coastal Plain

35. Research Questions
How do rates of denitrification vary in the Cahaba River?
2) How does denitrifier community composition change over space and time?
3) How does denitrifier activity and abundance change over space and time?
Denitrification in streams receiving large inputs of NO3- is limited by organic carbon lability.
Primary producer distribution will control denitrification longitudinally.
Seasonal changes in labile OC availability will affect rates of denitrification.

37. Testing for C and N Saturation
Coastal Plain Site 2 – Dec 2010
Denitrification Potential (nmol N2 g-1 hr-1)
NO3- Concentration Added (mmol)

38. Testing for C and N Saturation
Fall Line Site 2 – Dec 2010
Denitrification Potential (nmol N2 g-1 hr-1)
NO3- Concentration Added (mmol)

39. -Delivery of NO3- to subsurface influenced by geomorphology (Böhlke et al. 2009)
-Competition with in-stream primary producers

40. Nitrogen and Carbon Response*
Early Summer *
Fall Line Late Summer
DNP (nmol N2 g-1 hr-1)
No significant response to nitrogen or carbon (p=0.05)
Late Summer
FL1: Middle of Fall Line, macrophyte-dominated shoals
F2: Bottom of Fall Line, transition to Coastal Plain
DNP (nmol N2 g-1 hr-1)
* Statistically significantly different from ambient denitrification potential rate; p=0.01, 0.02