From Physiology to Global Change: How Do Soil Bacteria Regulate Global Atmospheric Methane?

EPA Grant Number: U916141
Title: From Physiology to Global Change: How Do Soil Bacteria Regulate Global Atmospheric Methane?
Investigators: Engelhaupt, Erika D.
Institution: University of Louisville
EPA Project Officer: Graham, Karen
Project Period: January 1, 2003 through December 31, 2006
Project Amount: $102,000
RFA: STAR Graduate Fellowships (2003) Recipients Lists
Research Category: Academic Fellowships , Fellowship - Terrestrial Ecology and Ecosystems , Ecological Indicators/Assessment/Restoration


The objective of this research project is to use estimates of population size in conjunction with kinetic studies to look for evidence of adaptation to different methane (CH4) concentrations with depth in the soil. Additionally, I will attempt to analyze population diversity, as a correlate of adaptation, using biochemical and molecular genetic approaches. Soil bacteria that oxidize CH4 have been recognized only by their activity and have not been grown in culture. Although the process kinetics have been examined in part, we do not understand the physiology of the active bacteria. We do not know whether there are different species (genotypes) of CH4 oxidizers, how different populations might be distributed, or how populations might respond to changes in steady-state CH4 concentration. One data set indicates a possible increase in enzyme affinity for CH4 with soil depth, as indicated by a decrease in Km with depth. An increase in enzyme affinity would indicate an adaptation to increase uptake of CH4, but we cannot interpret a decrease in Km as an increase in specific affinity without knowing how population size changes concomitantly.

My research will address the following three questions and four hypotheses:

General Question: Do atmospheric CH4 oxidizers in soil use atmospheric CH4 for growth?

Research Question 1: Do atmospheric CH4 oxidizers adapt to decreasing CH4 availability with depth in the soil profile?

Hypothesis 1: Decreasing CH4 oxidation rates with depth reflect decreasing population size due to CH4 limitation.

Hypothesis 2: Specific affinity for CH4 increases with depth, reflecting adaptation to lower CH4 availability.

Research Question 2: Are atmospheric CH4 oxidizers diverse across different ecosystems and with depth in soil?

Hypothesis 3: The structure of phospholipid fatty acids (PLFAs) that incorporate 13CH4-C will vary across ecosystems and with depth in the soil.

Hypothesis 4: The number of particulate methane monooxygenase A (pmoA) gene sequences, as revealed by polymerase chain reaction/denaturing gradient gel.


My central strategy is to test the hypothesis that atmospheric CH4 oxidizers adapt to ambient CH4 concentrations in soil, pointing to the availability of atmospheric CH4 as a selective pressure on their physiological evolution. CH4 availability in soil is diffusion-limited and decreases with depth because of biological oxidation in overlying soils. Thus, if atmospheric CH4 oxidizers use CH4 for growth, their population size should decrease with depth. Although a correlation between population size and CH4 concentration alone would not prove substrate utilization, such a correlation must exist to support this hypothesis. Moreover, the size of the active population is necessary to evaluate specific affinity as evidence of adaptation. Specific affinity is the maximum unsaturated uptake rate per unit of biomass. Recent work has demonstrated a decrease in Km with soil depth, suggesting a possible increase in specific affinity. This observation, however, cannot be interpreted as an increase in enzyme affinity without knowledge of the relative population shift, because specific affinity equals Vmax/Km, and Vmax varies directly with biomass.

Adaptation to different steady-state CH4 concentrations could represent functional plasticity within a particular population, or it could represent a shift in dominance of different populations. I will examine population diversity by following labeled CH4 incorporation into phospholipid fatty acids (PLFA) and pmoA gene sequences encoding the enzyme for CH4 oxidation, particulate methane monooxygenase (pMMO).

Initial studies will be conducted using soils from the Bonanza Creek Experimental Forest (Alaska) and Harvard Forest Long-Term Ecological Research (LTER) sites. The biogeochemistry of these sites has been studied extensively, and atmospheric CH4 oxidation has been demonstrated and studied in both soils. The soils have been shown to maintain their atmospheric CH4 oxidation activity during long-term storage in the lab, which allows maintenance of a stable microcosm community for experimentation.

Each site has different atmospheric CH4 oxidation kinetics. In Bonanza Creek soils, atmospheric CH4 oxidation in deeper soils increased after exposure to higher CH4 concentrations, suggesting an enrichment effect. These soils also exhibited a starvation effect (decreased oxidation rates) when incubated without CH4. In contrast, Harvard Forest soils have not responded to either enrichment or starvation, yet their atmospheric CH4 oxidation rates are approximately ten times higher than those of Bonanza Creek soils, possibly due to their coarser texture. The active populations in these two soils may have different nutritional physiologies; perhaps some atmospheric CH4 oxidizers use CH4 for growth and some do not. These two sites will allow comparison of soils with different apparent atmospheric CH4 oxidation dynamics and provide a comparison of response in two different biomes—northern boreal forest and northeastern temperate forest.

I will use the number of PCR-amplified pmoA sequences evident as DGGE bands to estimate the number of active atmospheric CH4 oxidizing populations across soils and with depth. I will use newly published PCR primer sets to amplify pmoA sequences in soils from 13CH4 incubation experiments and separate bands representative of different genotypes using DGGE. If population growth is large, then genomic DNA of the active populations would by heavily enriched with 13C and could be separated from total community DNA by ultracentrifugation in a CsCl gradient. Such a separation would enhance the chances of specifically amplifying the pmoA gene of active populations. Selective sequencing of bands unique to a given soil depth or ecosystem will provide data on potential evolutionary divergence of physiologically adapted populations.

Supplemental Keywords:

fellowship, soil bacteria, atmospheric methane, methane monooxygenase A, PCR, oxidation kinetics, Bonanza Creek Experimental Forest, Harvard Forest Long-Term Ecological Research site, RFA, Scientific Discipline, Air, climate change, Air Pollution Effects, Environmental Monitoring, Atmospheric Sciences, Atmosphere, oxidation capacity, green house gas concentrations, soil bacteria, population abundance, atmospheric methane, environmental stressors, global warming

Progress and Final Reports:

  • 2003
  • 2004
  • 2005
  • Final