Grantee Research Project Results
Final 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 , Bohlke, J. K. , Smith, Richard L. , Revesz, K.
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 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 was 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, Massachusetts. First, oxygen concentration profiles and stable isotope ratios were used to infer the net effect of aerobic respiration on the aquifer scale. Second, natural gradient tracer tests were 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, were used for small-scale examination of the process. The estimates of rates and kinetic parameters by the latter methods were used to compare with the tracer test and isotope results. Based on the results from these approaches, the aquifer was characterized within the context of a subsurface ecosystem, integrating the combined effects of the hydrology, geochemistry, and microbiology on the process of oxygen consumption. It also was characterized by both vertical and horizontal gradients of dissolved oxygen, providing a range of electron acceptor demand from low in the uncontaminated zone of the aquifer, to moderate in the contaminated zone.The results have provided a better understanding of aerobic respiration as a process in the subsurface, facilitated interpretation of 18O2 natural abundance data from this and other field studies, and examined whether the respiration assays that were developed for other environments have utility for groundwater environments.
Summary/Accomplishments (Outputs/Outcomes):
Oxygen Profiles and Oxygen Isotope Ratios. Sharp vertical gradients of oxygen were evident within the groundwater contaminant plume on Cape Cod. Dissolved oxygen concentrations decreased with depth from levels near atmospheric equilibrium in shallow groundwater to anoxia in the core of the plume. There also was a relatively thick vertical interval in which oxygen concentrations persisted in the 1-10 micromolar (µM) range. The depth of the oxygen gradient sank relative to the water table as the contaminant plume moved down-gradient, but was evident even after several kilometers of transport.Microbial processes commonly fractionate low molecular weight molecules, resulting in an enrichment of the heavier isotopes in the substrate and lighter isotopes in the products. Fractionation of oxygen isotopes by aerobic respiration has been demonstrated in laboratory experiments and open oceans, but has not been examined previously in groundwater environments. The isotopic ratio of 18O/16O in dissolved molecular oxygen changed in the Cape Cod aquifer as a function of the oxygen concentration. As the oxygen concentration decreased, the oxygen became increasingly enriched in 18O; 18O values ranged from about +25 per mil (?) for near air saturation to +45 ?. These results are consistent with kinetic isotope fractionation by aerobic respiration. However, when fit to the Rayleigh fractionation equation, the apparent fractionation factors ( = -2 to -10 ?) are somewhat smaller than many values derived from closed-system experiments or field studies ( = -20 ?). This was due, in part, to dilution of oxygen via dispersion and diffusion during the reaction process.
Natural Gradient Tracer Tests. Intermediate scale, in situ assessment of aerobic respiration was accomplished using natural gradient tracer tests. The tests were conducted using 15-port multilevel samplers (MLS) to introduce a tracer into a selected location within the oxygen gradient. The tracer cloud was transported down gradient by natural groundwater flow and intercepted with rows of MLSs situated perpendicular to the water-table gradient. Sodium bromide was used as a conservative tracer to determine dispersion and advective transport and to track the path of the tracer cloud.
Aerobic respiration in the Cape Cod aquifer was assessed using two different types of tracers. When oxygen concentrations were relatively high (>30 µM), natural oxygen was replaced by exchange using 18O2 (98 atom percent) without altering the background concentration. For these tests, samples were collected and analyzed for the product of aerobic respiration, H218O. When the oxygen concentration was low (<20 µM), air was used to increase the dissolved oxygen concentration 20-80 µM above background.
For the air tracer tests, the rates of oxygen consumption were about 1 µmole (liter aquifer x day)-1 for 5.5 meters of transport. These rates must be considered a potential rate because the in situ oxygen levels were increased above background. However, other in situ conditions were not altered, so the rate is relevant within the context of organic carbon (i.e., available supply of electron donor), microbial populations, and hydrologic flow. The rate can be viewed as an estimate of Vmax for aerobic respiration within the 20 µM oxygen horizon and subsequently compared with the microcosm oxygen uptake results (see below). For the 18O2 tracer tests, enrichment of 18O in H2O was 0.1 ? in the breakthrough curves for two separate tests. These results indicated respiration rates 0.8 µmoles (liter aquifer x day)-1. The lower rates of oxygen consumption are consistent with the 18O2 test being conducted higher up in the oxygen gradient (i.e., less contaminated).
Microcosm Determination of Aerobic Respiration. Assessment of aerobic respiration from the small-scale (centimeter-scale) perspective was accomplished using laboratory incubations (microcosms) with freshly collected aquifer core material. A variety of incubation techniques have been developed for surface-water systems to measure oxygen consumption and heterotrophic activity for indigenous microbial communities. Two of these were adapted for use in this groundwater study.
The first method was the direct determination of the Michaelis Menten kinetic parameters for aerobic respiration using substrate depletion progress curbs. Aquifer core material was slurried with groundwater in sealed serum bottles, to which varying amounts of oxygen were added. The bottles were incubated with slow rotational mixing at in situ temperatures and oxygen concentrations monitored with time using gas chromatography. Oxygen uptake in these microcosm incubations was slow, but measurable. Kinetic parameters were determined using best-fit simulations with a fourth-order Runge Kutta numerical approximation of the Michaelis Menten equation. The shape of the oxygen uptake curve was dictated by the kinetic parameters for oxygen consumption. Because there was little chemical oxygen demand in these samples, all oxygen uptake was assumed to be due to aerobic respiration. When converted to comparable units, these microcosm rates are >100-fold higher than the tracer test rate estimates. Microcosm replication within a mixed core sample usually was very good, but there was considerable variability between cores. The latter reflects the heterogeneity within the aquifer on this small scale and the disruptive effects that obtaining the core material had upon the attached microbial communities.
The second microcosm incubation method used was the determination of bacterial ETS activity 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. ETS activity assays have been used almost exclusively in surface-water systems; little is known regarding the utility in groundwater. In this study, the reduction of tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), to INT-formazan was measurable in aquifer microcosms, indicating microbial respiratory activity. However, rates of aerobic respiration calculated from INT reduction were 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 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.
Conclusions. A multi-scale, integrated approach was the best choice for studying a microbial process in the saturated subsurface. This study demonstrated that aerobic respiration within an aquifer does influence the isotopic signature of molecular oxygen in groundwater. That signature is an integrated result of the processes influencing oxygen concentration along a flow path. More detail about the in situ function of the microbial process relative to transport can be obtained on an intermediate scale using natural gradient tracer tests. These tests provide the in situ quantification of the rates of oxygen consumption and a means to compare consumption to oxygen dispersion and diffusion across the oxygen gradient. Finally, mechanistic information such as the reaction kinetic parameters can be obtained using small-scale laboratory incubations. These incubations also enabled the assessment of the short-term responses of the groundwater ecosystem to environmental perturbations or changes in electron donor supply.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 14 publications | 1 publications in selected types | All 1 journal articles |
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Hatzinger PB, Palmer P, Smith RL, Penarrieta CT, Yoshinari T. Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria. Journal of Microbiological Methods 2003;52(1):47-58. |
R824787 (Final) |
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Supplemental 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.