Students in my lab are involved in microbial community analysis of wetland sediments and community level analysis of nitrate metabolism in freshwater wetlands.

Wetlands

Community level analysis of nitrate metabolism in freshwater wetlands

Wetlands are critical ecosystems for not only many plants and animals, but also for humans. In these soggy pieces of land, many of the chemicals we introduce into the environment are transformed into less toxic substances. Perhaps the most important biochemical process occurring in wetlands is denitrification.

Runoff from fields, lawns and even road surfaces transports an excess of nitrogen into nearby streams. This nitrogen isn't the inert nitrogen gas found in our atmosphere, but rather nitrogen in the forms of nitrate, NO₃^1-, nitrite, NO₂^1-, and ammonium, NH₄¹⁺, that are found most often (but not exclusively) in fertilizers. Just as these substances can turn your yard a lovely green, so too can they feed algae in various bodies of water. Algae blooms block sunlight to water plants growing beneath the water's surface. When the algae die, the decay process consumes oxygen, producing a condition known as hypoxia, which means the water has too little oxygen for organisms such as fish and crabs. However, the microorganisms in wetlands surrounding our waterways can convert these excess nutrients into gasses that escape into the atmosphere. Hence, by preserving wetlands, we can reduce our impact on our waterways. Currently both the Gulf of Mexico and the Chesapeake Bay suffer from hypoxic conditions which produce regions known as “dead” zones.

Click here to go to NOAA’s website on the Gulf of Mexico’s hypoxic zone

Click here to go to the Chesapeake Bay Foundation’s fact sheet.

Some of the biochemical reactions taking place in wetland soils are:

Ammonia oxidation: 2NH₄¹⁺ + 3O₂ → 2NO₂^1- + 2H₂O + 4H¹⁺

Nitrite oxidation: 2NO₂^1- + O₂ → 2NO₃^1-

Denitrification: NO₃^1-+ 2H¹⁺+ 2e^1- → NO₂^1- + H₂O

NO₂^1- + 2H¹⁺ + 1 e^1- → NO + H₂O

2 NO +2H¹⁺ + 2e^1- → N₂O + H₂O

N₂O + 2H¹⁺ + 2e^1- → N₂ + H₂O

There are two forms of the denitrification pathway: dissmilatory and assimilatory. Dissmilatory denitrification uses nitrate as an alternative to oxygen for the electron acceptor in the energy producing electron transport system. Assimilatory denitrification uses the denitrification pathway with an alternative set of enzymes to reduce nitrate to ammonia for the bacteria's biosynthetic pathways. This process reverses the ammonia oxidation reaction and thus requires some energy input. Dissmililatory denitrification produces the gasses NO, N₂O (a greenhouse gas), and N₂ whereas the assimilatory process does not produce gasses as a by-product.

Research Goals:

1.  To study the effect of seasonal temperature change on the overall rate of denitrification which may be useful in nitrogen management plans for farmers and lawn service providers.

2.  To find one or more microbial genetic markers in soil samples that can be detected through simple, inexpensive soil tests so that developers can determine how close to a wetland site they may build without actually disturbing the wetland. It is my hope that with the means to accurately determine where the functional edge of a wetland is, both environment preservation and economic development can coexist.

Research Approach:

Goal 1: Denitrification as a biochemical process can be followed in a number of ways. For this project, we are using an ion selective nitrate probe to follow the reduction of nitrate by samples of wetland sediments obtained at different times of the year. In addition, since the overall process of dissimilatory denitrification produces the gasses NO, N₂O and N₂, we can follow gas production via a sensitive pressure probe and gas chromatography. With these two measures, we should be able to determine the metabolic participation of the dissimilatory pathway vs the assimilatory pathway.

Goal 2:  Microbial DNA can be used to analyze, and even identify, specific members of the microbial soil community. Microbial genomic DNA can be extracted from soil samples then subjected to PCR (polymerase chain reaction). By designing different PCR primers, the whole community can be analyzed or a subset of the community can be analyzed. Since I'm most interested in the dentrification process, PCR primers that amplify parts of the narG (dissimilatory nitrate reductase, subunit G), nap A (a second dissmilatory nitrate reductase, subunit A) and nirK (assimilatory nitrate reductase, subunit K) genes can be used to create a community profile that focuses on denitrification. When cloned and sequenced, these PCR products can be used to identify the bacteria involved.

Microbial Community Analysis of Wetland Sediments

(in collaboration with the Geography program)

This new project will be analyzing sediment samples from 6-8' cores taken from several local water retention ponds. We will be looking at how the microbial community varies with depth and possibly over time by using RFLP analysis and DNA cloning/sequencing of the 16s ribosomal genes isolated from microbial DNA extracted from these sediments. We may also examine how the various nitrate processing genes also vary with depth in these samples. The research approach will be similar to goal 2 of the project described above.

Page updated: August 14, 2014

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