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