exploring community dynamics of denitrifying bacteria in the cahaba river
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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. Effects of N-loading. Acidification/ eutrophication of freshwater systems - PowerPoint PPT PresentationTRANSCRIPT
Exploring Community Dynamics of Denitrifying Bacteria in the Cahaba River
Corianne Tatariw, Elise Chapman, Dr. Jennifer EdmondsDepartment of Biological SciencesUniversity of Alabama, Tuscaloosa
Effects of N-loading Acidification/
eutrophication of freshwater systems
Loss of biodiversity
Images from NOAA
•Harmful algal blooms•Eutrophication/ hypoxia in coastal systems
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?
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)
NO3- NO2
- NO N2O N2
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
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
THE CAHABA RIVER
Valley Ridge (VR)
Fall Line (FL)
Coastal Plain (CP)
VRFL
CP
VR-1VR-2
FL-1FL-2
CP-1CP-2
Poro
sity
0.2
0.4
0.6
0.8
POROSITY IN THE CAHABA
NO
3-N
(ppb
)
0
500
1000
1500
2000
2500
3000
Birmingham
THE CAHABA RIVER: A NITRATE SINK
SRP
(ppb
)
0
50
100
150
200
250
300
350
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
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
ANOSIM: nirS No significant
difference by geophysical province or date
nirS dominates in anoxic environments which may have less variable conditions Valley Ridge
Fall LineCoastal Plain
Global R = 0.157p = 0.03
2D Graph 2
X vs Y - CP X vs Y - FL X vs Y - VR
2D Stress = 0.14
ANOSIM: nirK
Global R = 0.223p = 0.001
nirS
Coastal PlainFall LineValley Ridge
Valley RidgeFall LineCoastal Plain
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
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
Methods Sediment samples collected with
microbial samples
Acetylene block method to measure denitrification potential rates
VR FL CP0
5
10
15
20
25
30
Apr Amb Jun Amb Sept Amb Dec Amb
Den
itrifi
catio
n P
oten
tial R
ate
(nm
ol/g
/hr)
DENITRIFICATION POTENTIAL
RATESD
enit
rific
atio
n Ra
te (
mmol
N2 g-
1 hr-1
)
a
abab
bp <0.05
VR FL CP0
5
10
15
20
25
30
SpringEarly SummerLate SummerWinter
ImplicationsThe Cahaba River is a
sink for NO3-
Lack of relationship between changes in community composition and denitrification rates suggests functional redundancy
AcknowledgementsCommittee Members
Dr. Jennifer EdmondsDr. Robert Findlay
Dr. Lisa DavisLab MembersElise Chapman
Courtney DragiffSusan Jozefiak
Ben Wilson
Field and Lab HelpChau Tran
Julie JarniganDiane Schneider
Marie Wilson
ANOSIM:16S rDNA at Fall Line 1
2 June 201024 June 201014 July 2010
2 June 201024 June 201014 July 2010
Global R = 0.414
p = 0.001
2D Stress = 0.16
14 July 2010
2 June 2010
24 June 2010
• Bacterial community structure changes over time through macrophyte growing season
Nitrogen and Carbon Response: Winter/Spring
VR FL CP0
2
4
6
8
10
12
14
16
DNP
(nm
ol N
2 g-1
hr-
1 )
DNP
(nm
ol N
2 g-1
hr-
1 )
VR FL CP0
5
10
15
20 *
* Statistically significantly different from ambient denitrification potential rate; p=0.05
VR FL CP0
10
20
30
40
50
Ambient500 mM N500 mM N/ 2500 mM C
Dec. 2010
Apr. 2011
The Cahaba River: An N Sink?
Fall Line
Humans and Nitrogen
Estimated deposition of reactive nitrogen in a) 1890s and b) 1990s
Galloway et al. 2004. Biogeochemistry 70: 153-266
• Fertilizer• Leguminous crops• Livestock • Fossil fuel
combustion
Microbially Mediated N-removal
Burgin and Hamilton. 2007. Front Ecol. Environ. 5: 89-96
Nitrite Reductase Functional Genes
nirS: Consistently anoxic environments, always active
nirK: Areas of DO flux, more active at night
Copper-containing nitrite reductaseNojiri et al. 2007. PNAS. 104: 4315-4320.
Nitrite reductase cytochrome-cd1Nurizzo et al. 1998. Biochemistry 37:
13987-13996.
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
C and N Limitation – June 2010
VR1 VR2 FL1 FL2 CP1 CP20
10
20
30
40
50
Ambient+N (500 mM) +N+C (500/2500 mM)
DN
P (n
mol
N2O
/g/h
r)
Site
VR1 VR2 FL1 FL2 CP1 CP20
10
20
30
40
50
Ambient+N (500 mM) +N+C (500/2500 mM)
DN
P (n
mol
N2O
/g/h
r)
Site
Den
itri
ficat
ion
Pote
ntia
l (nm
ol
N2 g-
1 hr-1
)
C and N Limitation – Sept 20102D Graph 5
VR1 VR2 FL1 FL2 CP1 CP20
10
20
30
40
50
60
Sept 2010 Amb Sept 2010 +N Sept 2010+C+N
DN
P (n
mol
N2O
/g/h
r)
Site
**
Significantly different from ambient DNP rate; p <0.05*
**
**
VR1 VR2 FL1 FL2 CP1 CP20
10
20
30
40
50
Ambient+N (500 mM) +N+C (500/2500 mM)
DN
P (n
mol
N2O
/g/h
r)
Site
Future Work1) 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?
1) Denitrification potential rate measurements in April 2011 will be highest at the Fall Line2) 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.
Date Site Avg Ambient StDev Temperature °C Porosity StDev AFDM StDevJun 2010 LB 8.161998949 0.332226 29.2 0.386133333 0.006886 2.601236945 0.377843
HL 6.289706525 0.641923 29.5 0.373494737 0.018102 2.283198858 0.254031CV 3.162459464 0.686203 29 0.3837 0.014843 1.740152112 0.08738SP 4.703626103 3.821566 29 0.357856642 0.009515 0.386379651 0.085188MJ 4.994243446 0.058324 0.392133333 0.020305 0.633802713 0.624277
03 Jun 2010 PP 18.83327397 7.527388 28.924 Jun 2010 PP 26.41588141 3.552288 30.1 0.478411111 0.057839 3.764592511 1.41164114 Jul 2010 US 6.471659572 1.451805 30.7
MB 4.279251108 1.649617SC 4.420255345 1.476759DD 4.165626458 1.037914
Aug 2010 PP 8.372572675 3.651942 32.2Sep 2010 PP 11.56170254 12.80802 28.8Sep 2010 LB 5.767593586 1.34986 0.38225 0.00916 3.987856567 0.847384
HL 7.111404042 0.805633 0.38875 0.365 2.071134073 0.239217CV 10.28448042 4.559856 0.382 0.007104 1.559518416 0.12772SP 5.749148825 0.785916 0.3175 0.00952 0.281233063 0.199009MJ 1.982245124 0.201137 0.37925 0.030766 0.410048422 0.369577
Dec 14 2010 LB 8.917669919 2.055431 4.5 0.286 0.069527 3.806077359 0.235186HL 6.862733321 1.916889 6 0.2612 0.056908 2.829575575 0.736192PP 18.48504912 2.247254 6.1 0.219333333 0.032934 4.112442571 0.405341CV 6.692058788 1.784798 0.401066667 0.028381 2.034080692 0.6658SP 6.893814842 6.023206 6.7 0.588266667 0.019129 3.732548565 1.639047MJ 7.50694151 0.692246 6.5 0.682933333 0.026544 3.302282683 0.318121
Click icon to add picture•Large and unregulated•North of Birmingham to Selma, AL•Confluence with Alabama River•Valley Ridge, Fall Line, Coastal Plain
Research Questions1) 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.
Testing for C and N SaturationCP-2 December 2010
0 100 200 300 400 5005
10
15
20
No CarbonCarbon
DN
P (n
mol
N20
/g/h
r)
[NO3-]
Den
itri
ficat
ion
Pote
ntia
l (nm
ol N
2 g-
1 hr-1
)
NO3- Concentration Added
(mmol)
Coastal Plain Site 2 – Dec 2010
CP-2 December 2010
0 100 200 300 400 5005
10
15
20
No CarbonCarbon
DN
P (n
mol
N20
/g/h
r)
[NO3-]
0 100 200 300 400 5003
6
9
12
NO3- vs DNP no C NO3- vs DNP +C
DN
P (n
mol
N20
/g/h
r)
[NO3-]
FL-2 December 2010D
enit
rific
atio
n Po
tent
ial (
nmol
N
2 g-
1 hr-1
)
NO3- Concentration Added
(mmol)
Fall Line Site 2 – Dec 2010
Testing for C and N Saturation
CP-2 December 2010
0 100 200 300 400 5005
10
15
20
No CarbonCarbon
DN
P (n
mol
N20
/g/h
r)
[NO3-]
› -Delivery of NO3- to subsurface influenced by
geomorphology (Böhlke et al. 2009)› -Competition with in-stream primary producers
Nitrogen and Carbon ResponseEarly Summer
X Data
VR FL CP0
10
20
30
40
VR FL CP0
10
20
30
40
50
Ambient500 mM N500 mM N/ 2500 mM C
VR FL CP0
10
20
30
40
50
Ambient500 mM N500 mM N/ 2500 mM C
Late Summer
DNP
(nm
ol N
2 g-1
hr-
1 )
DNP
(nm
ol N
2 g-1
hr-
1 )
No significant response to nitrogen or carbon (p=0.05) FL1 FL2
0
10
20
30
40
50
60
Col 27 Col 29 Col 31
**
* Statistically significantly different from ambient denitrification potential rate; p=0.01, 0.02
Fall Line Late Summer
FL1: Middle of Fall Line, macrophyte-dominated shoals
F2: Bottom of Fall Line, transition to Coastal Plain