Projects presently ongoing in our laboratory

Aluminum Control of Organic Carbon Cycling in Temperate Forest Ecosystems
The release of dissolved organic matter (DOM) from forest floor material constitutes a significant flux of C to the mineral soil in temperate forest ecosystems, with estimates on the order of 120 to 500 kg C ha-1 year-1.  Interaction of DOM with minerals and metals results in sorptive fractionation and stabilization of OM within the soil profile.  In particular, aluminum (Al), which derives from crustal rock, has been implicated recently as exerting significant control over soil C cycling in a range of ecosystem types, but the underlying mechanisms of stabilization - which involve tight coupling among chemical, mineralogical and microbiological processes - are not well known.  We hypothesize that Al-DOM complexation impacts the physical and chemical properties of DOM and, in turn, its sorption and biodegradation.  Further, we postulate that in a given ecosystem type (and therefore with climatic variables fairly constant), differences in C storage are controlled largely by the type of parent rock.
Pinus ponderosa forests, established on basalt, granite and limestone parent materials, are being used as model ecosystems for the proposed work. Characterization of soil mineral assemblage, litter quality, soil C MRT, and Al-tolerant microbes is being coupled with lab-based experiments on soils collected from the sites to quantify Al-DOM complexation, adsorption and biodegradation.  Shifts in microbial community with increasing Al-DOM complexation will be quantified via DNA analysis of the microbes degrading 13C labeled Al-DOM complexes in soil microcosms. 

Can soil genomics predict the impact of precipitation on nitrous oxide (N2O) fluxes from soil?
Nitrous oxide (N2O) is a potent greenhouse gas and presently emissions from soils are so poorly understood we can not predict how changes in precipitation will impact the release of this important greenhouse gas. Most nitrous oxide is released through biological processes, especially nitrification and denitrification.  These processes not only affect the release of greenhouse gasses to the atmosphere but also impact important ecosystem services such as purifying air or water and regenerating soil fertility.  Soil genomics, the analysis of nucleic acids from soil, can provide the basis for new ecosystem models by identifying marker sequences that estimate the impact of our energy use on N2O fluxes from soil.

15N Natural Abundance of Soil Microbial Biomass as a Tool for Assessing Controls on N-cycling Processes in Ecosystems
 Nitrogen limits plant growth in many soils around the world.  Plants compete with microorganisms in the soil for nitrogen.  When there is lot of carbon, in the form of organic matter or dead organisms, and relatively little nitrogen the microorganisms will take up most of the available nitrogen.  In contrast, when microbial growth is limited by carbon, soil microorganisms will increase the amount of nitrogen available to plants by breaking down organic matter that contains nitrogen.  At present it is difficult to measure if soil microorganisms are competing with plants for nitrogen or if they are helping plants to grow by supplying nitrogen.
Nitrogen atoms occur in a least two forms in the environment. Most of the atoms have 7 protons and 7 neutrons (14N) but a few atoms will consist of 7 protons and 8 neutrons (15N).  We have found that soil organisms are enriched in 15N relative to nitrogen in the soil solution and propose that the ratio of 15N to 14N in the microbial biomass indicates if microbial growth is limited by nitrogen or by carbon and that therefore it is possible to ascertain if plant growth is promoted or restricted by soil organisms.

Relationship between natural abundance δ15N of DNA
extracted from soil and abundance of archaeal amoA genes in soil.

Ammonia oxidizing archaea in semi arid soils
Autotrophic ammonia-oxidizing communities, which are responsible for the rate-limiting step of nitrification in most soils, have not been studied extensively in semi-arid soils. We have been measuring abundances of soil archaeal and bacterial amoA with real-time PCR in semi-arid ecosystems along an elevation gradient in northern Arizona. Archaeal amoA is the predominant form of amoA at all sites, however archaeal amoA to bacterial amoA ratios range from 17 to more than 1600. Though size of ammonia-oxidizing bacteria populations are correlated with precipitation, temperature, percent sand and soil C:N; we have not uncovered significant relationships between ammonia-oxidizing archaea populations and any of the environmental parameters evaluated in our studies. Our results suggest that in these soils archaea may be the primary ammonia-oxidizers, and that ammonia-oxidizing archaea and ammonia-oxidizing bacteria occupy different niches.
Abundance of bacterial (open bars) and archaeal (closed bars) amoA genes
in soils from five different northern Arizonan ecosystems during two different
seasons. Error bars show standard errors of the mean and letters denote
significant differences among amoA abundances as determined through Tukey
analysis (α = 0.05).

Characterization of growing microorganisms in soil through stable isotope probing with H₂¹⁸O.
We developed a new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H₂¹⁸O.  To test if sufficient amounts of 18O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing (SIP), Escherichia coli DNA was labeled by cultivating the bacteria in Luria broth with H₂¹⁸O and labeled DNA was separated from [16O]DNA on a cesium chloride gradient.    Soil samples were incubated with H₂¹⁸O for 6, 14 or 21 days and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days indicating that 18O can be used in stable isotope probing of soil samples.  Labeling soil DNA with H₂¹⁸O allows identification of newly grown cells.  In addition, cells that survived but did not divide during an incubation period can also be characterized with this new technique because their DNA will remain without label. 

Biodiesel production from micro algae
Among the most important challenge facing the Unites States in the 21st century is the procurement of a dependable, environmentally friendly source of energy.  Present energy supplies are dependent on fossil fuels, especially oil, which are predominantly located in regions outside the United States and which upon incineration release carbon dioxide, a greenhouse gas.  Certain microalgae, such as the diatom Cyclotella cryptica convert up to 50% of their biomass into lipids which may, in turn, be transformed to biodiesel through a simple chemical process.  Biodiesel can be used to power diesel engines in cars or to fuel mobile diesel powered electricity generators. We have been studying Cyclotella cryptica and have recovered a strain that can grow rapidly on a cheap medium we have formulated.  In addition we discovered that lipid accumulation by this strain is strongly impacted by the salinity of the medium.

A lipid accumulating diatom isolated from Rio de Flag wetland in Flagstaff AZ.
Light microscopy image of isolate stained with the dye Nile Red is shown in A.
In panel B a fluorescence microscopy image of the same sample is shown. In
a non-polar environment Nile Red fluoresces yellow indicating the diatoms are
filled with lipids.

Adaptation and Implementation of a water quality curriculum in BIO 369 Environmental Microbiology
One of the most important environmental issues facing the Southwestern US is the availability and quality of water.  Water treatment is therefore an increasingly important technology at the forefront of environmental microbiology.  We will adapt and implement a new water quality curriculum, developed with funds from Arizona state proposition 301, in an environmental microbiology course to better prepare undergraduate microbiology students to face this challenge. In addition, we will engage high school and community college students from around Northern Arizona in water quality laboratory exercises.
The field of environmental microbiology has exploded in recent years, merging molecular with traditional approaches to address the complex roles of microorganisms in serious environmental problems. Rigorous undergraduate training in this area requires hands-on experience with multiple approaches to address real-world issues. The purpose of the laboratory course in environmental microbiology at Northern Arizona University (NAU) is to expose students to various aspects of this field in order to acquaint them with key questions and methodologies currently driving the field.
We have  implemented a laboratory curriculum focused on water quality microbiology by linking field experiences at the nearby Rio De Flag Water Reclamation Facility with current molecular and functional laboratory approaches. Students will acquire treated and untreated water samples from this facility for determination of abundance of viruses and organic pollutants.  Student teams will also design, conduct, and present experiments focused on the removal of viruses or pollutants using samples from the reclamation facility.
 
Students from Shonto Preparatory Technical School
visiting NAU and using electrophoresis equipment
purchased with funding from an NSF-CCLI grant.