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Research Team
Research Team
Olivia Pohl
Olivia Pohl
Third year microbiology major studying ammonia oxidizing bacteria in Dr. Bollmann's lab.
Introduction
Introduction
Ammonia oxidizing bacteria (AOB) play a critical role in the global nitrogen cycle through the oxidation of ammonia to nitrite. This process is essential in both natural ecosystems and engineered systems such as wastewater treatment plants, where AOB contribute to the removal of human-derived ammonium.
AOB frequently experience environmental stress, including both elevated and limited ammonia availability. High ammonia concentrations can inhibit growth and disrupt cellular metabolism.
Ammonia oxidation in AOB is catalyzed by ammonia monooxygenase (AMO), a membrane-associated enzyme complex encoded by the amoCAB operon. Preliminary research has shown the single copy amoC gene of Nitrosomonas eutropha to be involved in stress response.
Understanding how AOB tolerate high ammonia stress and recover following exposure is crucial for predicting the stability of nitrifying populations under fluctuating environmental conditions. Improved understanding of AOB stress tolerance mechanisms may help predict nitrifier responses to increasing nitrogen inputs and inform strategies to improve the stability of engineered nitrification systems.
Research Questions
Research Questions
How does increasing ammonia concentration influence growth and amoC transcription in N. eutropha, and is amoC expression consistent with a stress-adaptive role under elevated ammonia concentrations?
Methods
Methods
Cells are grown at 1µM to 500µM ammonia concentrations.
Growth rates of the cultures are determined using nitrite measurements.
Cells are filtered from the culture and RNA is isolated.
DNase treatment and reverse transcription are performed to obtain mRNA.
amoC single copy and operon mRNA are quantified using RT-qPCR.
Data analysis is performed to determine the impact of high ammonia concentrations on the amoC genes.
Results and Discussion
Results and Discussion
Results
Results
Figure 1. The change in transcription of single copy amoC and amoCAB operon under varying ammonia concentrations.
Discussion
Discussion
Preliminary transcriptional analysis suggest that the single-copy amoC gene in N. eutropha shows a stronger increase in expression at elevated ammonia concentrations compared to the amoCAB operon. This patterns suggests that the single-copy amoC may play a distinct role in the cellular response to high ammonia stress, potentially contributing to maintenance of ammonia monooxygenase activivity under inhibitory conditions. To further investigate this response, ongoing work will examine amoC transcription across a finer gradient of ammonia concentrations using RT-qPCR. These experiments will help determine whether the observed expression differences reflect a consistent stress-responsive pattern under elevated ammonia conditions.
Future directions
Future directions
Future work will examine amoC transcription under additional stress conditions relevant to engineered nitrification systems, including exposure to reactive oxygen species, increased nitrite concentrations, and changes in pH.
Expression of other stress-responsive genes will also be evaluated to better understand the transcriptional responses of N. eutropha associated with AOB during environmental stress.
Acknowledgements
Acknowledgements
This work was completed under the mentorship of Dr. Annette Bollmann. I would also like to thank Dr. Sabita Ghimire for her preliminary RNA sequencing work on N. eutropha, which provided the foundation of the experimental direction of this study.
References
References
Berube PM, Stahl DA. 2012. The divergent AmoC3 subunit of ammonia monooxygenase functions as part of a stress response system in Nitrosomonas europaea. Journal of Bacteriology 194(13):3448–3456. doi:10.1128/JB.00133-12.
NACE Career Readiness Competencies
NACE Career Readiness Competencies
I have gained communication skills by working with my mentor and other scientists to complete this project.
I have gained critical thinking skills surrounding forming and carrying out experiments.
I have learned to use technology to carry out data analysis and take away conclusions from my results.
I have gained laboratory skills and experience strengthening my applications to graduate school.
Research Compliance Protocols
Research Compliance Protocols
All work was done in compliance with IBC protocol.
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