Tracking C Flow during Methane Oxidation into Methanotrophs using 13C-PLFA Labeling in Pulsing Hydrologic Freshwater Wetlands
Funding Agency: USEPA
Collaborator: William Mitch
Ph.D. Student: Taniya Roy Chowdhury
Microbial Ecology of Methanotrophs in Pulsing Wetland
Wetlands not only offer a means to offset the contributing factors to climate change, but can also be a significant contributor to greenhouse gas emissions. Methane (CH4) in soils is a key regulator of the atmospheric concentration of this important trace gas. It is estimated that 25% of the total CH4 is caused by wetlands. This is caused by anaerobic conditions that enable methanogens to produce CH4. However, the net flux methane is controlled by microorganisms that oxidize atmospheric CH4, methanotrophs. Although work in pure cultures and lab incubations have shown the potential importance of methanotrophs in regulating net CH4 flux, few field based studies have been done on the microbial ecology of these CH4 oxidizing communities and how they respond to differing land management systems. For example, there is considerable interest in enabling current wetlands and creating wetlands that have hydrologic pulsing that is driven by seasonal rainfall and watershed dynamics. However, how little is known as to how this might affect net CH4 emissions. A major limitation to study methanotrophs is adequate methods that connect them specifically to CH4 oxidation.
Isotope-labeling techniques such as stable-isotope probing (SIP) and PLFA-labeling offers potential to overcome classical microbiological methods for studying the methanotrophs relative to CH4 oxidation. This approach is a significant advancement as it is a cultivation-independent technique. The main objective of this study is to see any differences in methane emissions with seasonal variations across a landscape gradient of upland soil, a pulsing zone and permanently flooded sites. Hydrologic pulsing in wetlands should lead to lower CH4 emissions. Nitrogen (N) gradients are expected to exist in all of these wetlands from inflow to outflow, because of nutrient inflows. Wetlands are also significant sinks of nitrate-nitrogen, particularly through denitrification. An inverse pattern of methane generation and nitrate-nitrogen concentrations in the wetlands in hydrologically pulsed wetland areas should be observed. Our overall study will emphasize the importance of environmental factors (e.g., air, water, and soil temperature), microbiology, and hydrology (seasonal pulsing) on methane oxidations. This work is being done at the Ohio State University, Olentangy Wetland Research Park.
In addition controlled lab studies will investigate the relative importance of nitrate-N gradients in the wetlands, particularly where such gradients exist for denitrification “competing” with methanogenesis in the oxidation of organic C in anaerobic conditions. We will use isotopic tracking of 13CH4 into methanotrophs and other microorganism across regional sites, and along redox and N gradients (upland, pulsing interface, and flooded landscape positions in wetlands) to understand the microbial mechanisms that control methane emission of flow-through wetlands.
Stable Isotope Probing: The recent development of isotope-labeling techniques such as stable-isotope probing (SIP) and PLFA-labeling has been a significant advancement in cultivation-independent techniques for studying the microbial ecology of methanotrophs. These techniques use atoms that are isotopically enriched (99% 13C) when compared with their natural abundance (1.1% 13C), which enables the fate of a compound to be followed under conditions that approach those occurring in the environment. Furthermore, by following incorporation of the enriched-isotope into microbial cells or cellular biomarkers, it is possible to identify directly the distinct microbial groups that actively incorporate a substrate.
The active methanotrophs in soils and sediments have been identified using either radioactive 14CH4 or stable 13CH4 isotopic forms of CH4 to label PLFAs. Following gas chromatography and individual-compound isotope mass spectroscopy (GC-IC-IRMS), C16 lipids extracted, isotopically enriched 13CH4 methanotrophs can be labeled with 13C track C flow through these populations. SIP is a powerful technique suitable for cultivation-independent identification of the methanotrophs that are active in situ. SIP.