Grantee Research Project Results
1998 Progress Report: In Situ Assessment of the Transport and Microbial Consumption of Oxygen in Groundwater
EPA Grant Number: R824787Title: In Situ Assessment of the Transport and Microbial Consumption of Oxygen in Groundwater
Investigators: Yoshinari, Tadashi , Smith, Richard L.
Current Investigators: Yoshinari, Tadashi , Bohlke, J. K. , Smith, Richard L. , Revesz, K.
Institution: The State University of New York , United States Geological Survey
Current Institution: New York State Department of Health , United States Geological Survey
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1995 through October 1, 1998 (Extended to September 30, 1999)
Project Period Covered by this Report: October 1, 1997 through October 1, 1998
Project Amount: $350,000
RFA: Water and Watersheds (1995) RFA Text | Recipients Lists
Research Category: Watersheds , Water
Objective:
The level of oxygen in groundwater is controlled by both the geochemistry and microbiology. Aerobic respiration, the microbial metabolic process that consumes oxygen, is fundamentally important to the overall functioning of an aquifer. The objective of this project is to investigate oxygen consumption on several different scales in parts of a large (>5 kilometers) plume of dilute sewage contamination in a sand and gravel aquifer on Cape Cod, MA. First, data from oxygen concentration profiles and stable isotope ratios will be used to infer the net effect of aerobic respiration on the aquifer scale. Second, natural gradient tracer tests will be used at an intermediate scale to measure in situ rates of aerobic respiration within different contours of the groundwater oxygen gradient. Third, two different types of laboratory incubations using aquifer core material, potential electron transport system (ETS) activity and oxygen uptake activity, will be used for small-scale examination of the process. The estimates of rates and kinetic parameters by the latter methods will be used to compare with the tracer test and isotope results.Progress Summary:
Our progress on this project is described in the following paragraphs:1. Oxygen Profiles and Oxygen Isotope Ratios
New Method. An improved method for analyzing isotopes of dissolved O2 in water samples was developed. In the previous method, samples were collected in evacuated vessels, from which the O2 was extracted under vacuum, purified by gas chromatography, and converted to CO2 for mass spectrometry. This procedure yielded precise analyses, but it required large samples (20 micromoles of O2) and included relatively complex procedures in the field and laboratory. The objectives of the new method were to simplify the procedures for sample collection and preparation and to permit analyses of smaller samples. In the new method, samples were collected in 150 mL serum bottles without headspace. Low-pressure headspace was created in the laboratory by extracting about 10 percent of the water, then aliquots of the headspace were extracted through a needle into a helium carrier gas, which led through a GC to the inlet of a Finnigan Delta Plus mass spectrometer operated in continuous flow mode. The isotopes in the O2 were measured by peak integration on masses 32 and 34 in headspace aliquots containing in the order of 1 micromole of O2, with reproducibilities around +0.3 per mil.
Natural Gradient O2 Isotopes and 18O2 Tracer Test. Calculations based on previous sampling indicate that there is an O2 deficit in the transition zone at the top of the plume that is consistent with the isotopic evidence for O2 consumption, but that the rate of vertical transport of O2 into the top of the plume is limited.
A second tracer test was conducted in which 18O-enriched O2 was introduced into the O2 gradient at the top of the wastewater plume. The *18O values of H2O during the Br breakthrough curve were altered by <0.1 per mil, similar to the results of the previous test. New estimates of O2 consumption rates based on dispersion/reaction calculations are approximately consistent with the low rates of reaction indicated by the tracer tests.
O Isotopes in Nitrate. Nitrogen isotopes in ammonium indicate that nitrification may be active near the upper boundary of an ammonium cloud in the wastewater plume under Ashumet Valley. Evidence for local nitrification, and its significance with respect to overall O2 consumption, was sought in the O isotopic composition of the nitrate in the same gradient. Results indicate that the O isotopes in the nitrate are not locally anomalous, possibly because nitrification is only a minor sink for the O2, or possibly because the nitrate and the ammonium are traveling at different rates, so they are not locally related.
2. Microcosm Determination of Aerobic Respiration
Additional core material was collected during Year 3 of this study from the sand and gravel aquifer at the Cape Cod site. Cores were collected above and within the oxygen gradient and the rates of oxygen consumption quantified. This was done under a series of differing initial oxygen concentrations to determine the microbial response to changing oxygen regimes and to calculate kinetic parameters for oxygen consumption.
Bacterial ETS activity was determined using tetrazolium salts. Tetrazolium salts act as artificial electron acceptors, preferentially intercepting electrons from a cell's electron transport system prior to molecular oxygen, thus functioning as a measure of aerobic respiration. It was found that the total ETS activity of bacteria in groundwater samples was 4- to 10-fold lower than rates obtained from the direct measurement of oxygen consumption in the laboratory microcosms. Additional studies with bacterial cultures isolated from the Cape Cod aquifer revealed that 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) was toxic to many groundwater bacteria. Thus, the low rates of aerobic respiration calculated from the INT assay probably reflect the toxicity of INT to some portion of the resident microbial population.
The results from further experiments revealed that tetrazolium salts of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) also are toxic to many groundwater bacteria at concentrations normally used for ETS assays, and may thus alter the composition of the microbial community under study. These results suggest that conclusions regarding respiratory activity and viable cell numbers in natural environments from tetrazolium salt assays should be made with caution.
Future Activities:
The following activities are planned:1. Using the new method for O2 isotope analysis, new sets of groundwater samples, which were collected in serum bottles from the O2 gradient at various positions along the top of the wastewater plume, will be analyzed to determine if there are systematic changes in the relative effects of dispersion and O2 consumption in the longitudinal (downgradient) direction along the top of the plume. Those results will be used to evaluate the long-term interactions between anoxic plume water and surrounding oxic recharge waters.
2. Further work is planned to investigate the O isotopes in nitrate sampled with the 18O-O2 tracer to seek evidence of nitrification by the tracer O2.
3. The results from microcosm incubations experiments for determining the oxygen uptake and data for aerobic respiration in the natural gradient tracer tests that were conducted in Years 1 and 2 of this project will be compared. This comparison will help determine which types of laboratory incubations, if any, best represent the in situ field experiments.
4. Additional studies will be carried out to better define the number and types of bacteria to which tetrazolium salts are toxic as well as those that do not incorporate the formazans during incubation.
Journal Articles:
No journal articles submitted with this report: View all 14 publications for this projectSupplemental Keywords:
groundwater, bacteria, aquatic, environmental chemistry, environmental biology, ecology, oxygen, aerobic respiration, northeast, Cape Cod, Massachusetts, MA., RFA, Scientific Discipline, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Water & Watershed, Bioavailability, Hydrology, Ecology, Chemistry, Bioremediation, Watersheds, fate and transport, microbial degradation, microbial consumption of oxygen, sodium bromide , electron acceptors, oxygen uptake kinetics, in situ bioremediation, tracer tests, aquatic ecosystems, sewage, bacterial degradationProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.