Dr. Miller’s Research Interests

 

Wetlands are critical ecosystems for not only many plants and animals, but also for humans.  In these sometimes tiny, soggy pieces of land many of the fertilizers, pesticides, herbicides and other chemicals we introduce into the environment get transformed into less toxic substances.

 

Wetlands come in many shapes and sizes.  Here on the Delmarva Peninsula, we have salt water, brackish water as well as fresh water wetlands.  Currently, my interests lie in using microbial biochemical and genetic markers to define the functional edges of wetlands.  I am working at two study sites as illustrated below.

 

 

My local wetland study site is in a conservation area near Salisbury’s mall.  This is a classic forested swamp that I am studying in order to determine if microbial genetic markers can be used to define the functional edges of a wetland..

 

 

               

 

Montandon marsh is a protected wetland in central Pennsylvania where I am currently involved in a collaborative study on wetland functions and microbial diversity.

 

Perhaps the most important biochemical process occurring in wetlands is denitrification.  Runoff from fields, pastures, and lawns transport an excess of ammonia, nitrate and nitrite into our lakes, rivers and bays.  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.  If we continue to destroy wetlands, our lakes, bays and even oceans may end up as deserts.  Currently both the Gulf of Mexico and the Chesapeake Bay suffer “dead” zones in which excess nutrients result in algae blooms.  When these algae die, the micro-organisms which consume them also consumes all of the oxygen so that no fish or other organisms can survive. 

 

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

                   2NH₄¹⁺ (aq) + 3O₂ (aq) → 2NO₂^1- (aq) + 2H₂O (l) + 4H¹⁺ (aq)

                   2NO₂^1- (aq) + O₂ (aq) → 2NO₃^1- (aq)

                   C₆H₁₂O₆ (aq) + 4NO₃^1- (aq) → 6H₂O (l) + 6CO₂ (aq) + 2N₂ (g)

 

          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.

          Click here to go to the EPA’s website on hypoxia in these and other areas of the US.

 

The goal of my research is to find microbial markers in soil samples that can be detected through simple, inexpensive soil test 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 the environment preservation and economic development can coexist.

 

Research approach for Salisbury Swamp project:

 

Once every week for an entire year, this swamp will be sampled at four sites.  Wooden pegs were driven into the ground to mark the sample sites so that each weekly sampling occurs at the same spots.  These four sites (see image below) are located in a forested upland position (peg 1), slightly upland from apparent normal water level (peg 2), slightly below the normal water level (peg 3) and in the center of the wetland (peg 4).

 

Schematic of Salisbury swamp sampling site. 

 

After collection, each sample is subdivided into three parts, one for determining moisture content and organic content, one for microbial community analysis based on extractable lipid profiles and one for microbial community analysis based on microbial DNA. 

 

The moisture content is determined by weighing out a wet soil sample then drying over night in an oven at 105°C.  The mass lost is due to water.  These dried samples are then placed into a muffle furnace and heated to ~450°C, hot enough to burn off most organic molecules.  The mass lost is mostly due to the loss of organics.  However, some lost mass is also likely due to the conversion of carbonates to oxides and carbon dioxide. 

 

 

Soil samples before carbon analysis (upper picture) and after ashing in a muffle furnace (lower picture).

 

 

Lipids are extracted from each sample using a Bligh-Dyer extraction buffer (containing 1:2:0.8 v/v/v chloroform:methanol:water).  The phases are split using a mild salt solution and the organic phase concentrated.  The ester-linked fatty acyl chains containing in this mixture are transmethylated using sodium methoxide and extracted using a hexane/chloroform solvent.  Those lipids entering the hexane phase are then analyzed using a gas chromatograph/mass spectrometer to create a lipid profile of the soil microbial community.  Comparison of these profiles using a simple Jaccard similarity index allows a quantitative comparison of the microbial community structure across time.

 

Two soil samples during extraction with the Bligh-Dyer solvent.

 

 

Soil lipid extracts before phase separation.

 

In addition to using microbial lipids as a biochemical marker for community structure, microbial DNA can be used to analyze, and even identify, members of the microbial soil community.  Microbial genomic DNA can be extracted from soil samples then subjected to PCR (polymerase chain reaction which copies small sections of DNA so that you have enough to analyze and clone).  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 and nirK 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. 

 

A DNA agarose gel of the narg soil community DNA fingerprint of four soil samples (lanes 1-4) and of the narg PCR products that were fingerprinted (lanes 6--9).  Lane 5 is a DNA sizing standard.

 

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