NC State Environmental Engineering Researchers Use Gene Probing to Improve Wastewater Treatment

Municipal and industrial wastewater has to be effectively treated to remove organic and nutrient pollutants before discharge to streams and rivers. Using leading edge technology, environmental engineers at NC State University are improving our understanding of how microorganisms can be influenced to increase the performance of wastewater treatment plants.

One major role for microorganisms is the multi-step conversion of harmful ammonia nitrogen to harmless nitrogen gas. According to Francis de los Reyes III, associate professor of Civil, Construction and Environmental Engineering, treatment plants typically supply large amounts of oxygen to allow microorganisms to convert ammonia to nitrite, then nitrite to nitrate. Nitrate is then converted to nitrogen gas by denitrifying bacteria. However, De los Reyes says less oxygen is required if the process is circumvented by bacteria that use nitrite instead of nitrate. Determining the identity and function of these nitrite-reducing bacteria is a challenge.

De los Reyes and NC State Ph.D. student Cesar Mota are studying this problem using a gene probing method that targets the messenger RNA of nitrite-reducing bacteria. Using a technique called Fluorescence In Situ Hybridization (mRNA-FISH), they are able to fluorescently tag active nitrite reducers in wastewater bioreactors. This technique targets the functional nitrite reductase gene, and helps them identify bacteria responsible for nitrite conversion.

“Targeting functional genes is a big leap in this research area. In previous methods, you had to know the identity of the organism before you can tag it. Now, you can target the gene and ask the question of which bacteria are performing the function,” de los Reyes said. “We are now extending the method to allow separation of the fluorescently labeled cells, and subsequent identification of the species involved. Once the organisms are identified, we can then track them and see which factors can be used to influence their growth, and ultimately, reactor performance.”

The new method, called Sequential mRNA FISH-Flow cytometry (SmRFF), also allows the researchers to spatially relate the different groups of organisms involved in the nitrogen conversions. Already, the researchers are seeing some interesting results, including some preliminary data showing that some ammonia oxidizing bacteria are also nitrite reducers.

“The finding is significant, since it means we can potentially run treatment plants with 60% less oxygen,” de los Reyes said. “The process will be best for wastewater with high nitrogen and low carbon, since you can also save potentially up to 100% of the carbon requirements.”

The current research is funded from a four-year National Science Foundation Microbial Interactions and Processes grant. Future applications of this work include targeting other functional genes important in environmental cleanup. “We can potentially use the new method to identify and quantify the microorganisms involved in cleaning up groundwater contaminated sites, or specific pollutants,” de los Reyes said. “Very few engineers are using molecular methods in this way.”

NC State University