Diversity and Functional
Variation of Denitrifying
Bacterial Communities in the
Cape Fear River Estuary
Brian Shirey
Marine Biology
Project Goals
• Provide insight into denitrifying
bacterial community structure
• Examine relationships between
bacteria and their environment
• Provide answers to questions
What’s The Big Deal About
Bacteria?

The good

The bad

And the ugly
Salmonella typhimurium
Denitrifying Bacteria

Anaerobic
– Utilize nitrate during respiration

Comprise over 50 different genera

Ubiquitous
Ecological Importance

Vital role in global nitrogen cycle
Ecological Importance cont.

Contribution to global warming
– Production of greenhouse gasses
NO3-
NO2-
N2O
NO
Nitrogen oxides
N2
Ecological Importance cont.

Effective in bioremediation
– Reduction of nitrate entering ground
and surface waters
Global Significance

Understanding the
mechanisms of
denitrification is critical
to our understanding of
the global nitrogen cycle

Applied methodologies
promote a healthy,
balanced, captive
ecosystem
Experimental Design

Collect samples

Perform bacterial diversity and
phylogenetic analysis

Perform community functionality
analysis
Sediment Sampling
Sediments collected
from 5 sites along
the cape fear river
estuary
Measuring Bacterial Diversity

DNA sequencing
– Isolation of functional genes encoding
for nitrate reductase

Comparative analysis
– Along environmental gradients

T-RFLP analysis
– Profile of community structure
Measuring Functional Diversity

Incubation experiments
– Designed to monitor denitrifying activity
Control
NaCl
Nitrate
Anaerobic Enrichment Cultures
Research Hypothesis
Different components of a community
will respond to different
environmental inputs to maintain
community functionality
Questions To Be Answered

Do denitrifying bacteria show the capability
to readily adapt to sudden changes in their
environment?

Is survivorship of denitrifying bacteria
greatly reduced in sub optimal conditions?

To what degree do environmental conditions
determine community structure?
Schedule

Phase 1
– Sequencing all DNA, setting up and
initialize incubation experiment
 Phase 2
– Completing incubation experiment
and performing T-RFLP analysis
 Phase 3
– Analyze data, write paper
Phase 1
Phase 2
Phase 3
Jan. Feb. March April May June July Aug. Sep. Oct. Nov. Dec.
Conclusion

Denitrifying bacteria have a tremendous
impact on the biosphere
 Our biosphere is fueled by an underlying
microbial world
 The global community and the microbial
community are eternally linked
 Bacterial community as a window into past,
present, and future
QUESTIONS?