Metabolic Gases in Aquatic and Marine Systems

Our Primary Citation

Our focus has been on measurements of metabolic gases in aquatic and marine systems owing to our academic background. We are not limited to water analyses, and our instruments can be readily configured for the sampling of dissolved gas streams or samples. There are over 300 published articles citing our original publication and the following applications come from that literature as well as selected non-published work.

Original Citation: Kana, T. M.; Darkangelo, C.; Hunt, M. D.; Oldham, J. B.; Bennett, G. E.; Cornwell, J. C. 1994. Membrane Inlet Mass Spectrometer for Rapid High-Precision Determination of N2, O2, and Ar in Environmental Water Samples. Anal. Chem. 66 (23), 4166-4170

Environmental Denitrification

Denitrification converts nitrate (NO3) to nitrogen gas (N2) and it is a critical process in the aquatic and marine nitrogen cycle because it removes nitrogen nutrient that would otherwise  degrade water quality. It is an anaerobic process typically  associated with sediments, hypoxic/anoxic water, groundwater and soils. The challenge has been to measure the very small amount of N2 from denitrification that is added to the high background concentration N2. In practice, measurements in natural environments typically require much better than 0.1% precision to detect Nfrom denitrification when working with hydric or water samples. This precision is not routinely attainable by gas chromatography, but is readily attainable by measuring N2/Ar ratios by mass spectrometry. Argon (Ar) happens to be a convenient basis (standard) for evaluating differences in N2 concentration because it is not biologically reactive.

Aquatic Ecosystem Metabolism and Oxygen Dynamics

Bay Instruments MIMS is an alternative to the previously preferred Winkler technique with the advantage of allowing smaller volume experiments and shorter incubation durations. Oxygen plays a critical role in ecological systems and its  concentration in water can range from zero to supersaturating. There are a number of suitable measurement techniques for dissolved oxygen (e.g. electrodes, Winkler, optodes), but for some environments (e.g. waters with low biological activity, such as the deep sea, open ocean or oligotrophic lakes), very high precision measurements are required to avoid overly prolonged incubations. This is analogous to the problem of measuring denitrification. MIMS offers comparable precision for O2/Ar as it does for N2/Ar (i.e. ~0.03%), which rivals the very best optimized Winkler method. MIMS can make these measurements on much smaller sample volumes than Winkler and is significantly quicker, however.

Sediment Studies

Isotope labeling studies  Another incubation method involves the application of isotopic (heavy) nitrogen (15N) as nitrate. Denitrification will lead to N2 containing the isotope. Denitrification, therefore, can lead to the production of three different masses of N2 (mass 28 as 14N14N, mass 29 as 14N15N, and mass 30 as 15N15N). The so-called 'isotope pairing technique' involves measuring the ratios of masses 28:29:30. This technique, originally developed using isotope ratio mass spectrometry, is now also accomplished using our MIMS.N2/Ar Method  Denitrification associated with sediment is commonly measured using sediment cores with overlying water. The water layer is subsampled, either through time using static core incubations, or by comparing inlet and outlet concentrations in continuously flowing chambers.  Both methods provide denitrification rate measurements. The static core method requires small volume samples to minimize dilution of the headspace water. In addition, the time-course incubations cause changes in O2 concentration, which may affect denitrification activity. Consequently, it is necessary to resolve changes in N2 concentration within a limited range of O2 concentration changes. Nominal precision on the order of 0.03% by our MIMS allows for such measurements.

Going to Sea

Our MIMS instruments have extensive experience operating aboard oceanographic ships. Prior to the founding of Bay Instruments, we were the first to take a MIMS to sea demonstrating the feasibility of getting data on station and in real time, which can have enormous scientific advantages. Mass spectrometers on research ships is fairly common today. There is current interest in understanding whether the oceans are, on balance, producing organic matter or consuming organic matter, of key importance to understanding the role of the oceans in climate change. Where the ocean is clear blue (same with lakes, for that matter), there are few organisms to affect the O2 and CO2 concentrations, and very high precision measurements are required. Our instruments have been used as an alternative to the previously preferred Winkler technique with the advantage of allowing smaller volume experiments and shorter incubation durations.

With modification of the cryotrap temperature, our instrument becomes a pCO2 analyzer which can provide simultaneous measurements of both dissolved O2 and CO2. Nutrient poor, low activity lake waters have been analyzed for CO2 and O2 dynamics using our high precision MIMS. We have to keep in mind that the MIMS method for CO2 does not include the hydrated forms, bicarbonate and carbonate ions, because charged molecules do not pass the membrane.

Open Water Studies

Although the core incubation methods are now standard approaches, open water sampling methods are actively being evaluated because this type of sampling approach integrates larger spatial and temporal scales. The challenge for these studies is to accurately determine the degree of disequilibrium in N2  or N2/Ar, relative to air saturation (i.e. how much additional N2 is in the natural water, relative to what would be there if no denitrification occurred). In many situations (flowing rivers, shallow lakes, etc), the disequilibrium is typically <1% relative to solubility, again requiring the need for high precision and a reliable method for standardizing against air solubility. Our method of preparing dissolved gas standards provides ~0.1% precision on gas (e.g. N2 or Ar) concentrations, which highly resolves temperature effects on solubility. Although these types of measurements are more challenging than for core incubation measurements, the MIMS method has opened new avenues for detecting denitrification in natural waters.

Aquatic Plant & Algae Photosynthesis: The Bubble Problem

Measuring aquatic plant photosynthesis can be problematic because oxygen concentration may rise to the level where small bubbles form on the leaf or on the sediment surface with benthic algae. Conventionally, researchers would either measure the dissolved O2 concentration changes, or the production rate of bubbles from cut leaves. It was not possible to measure photosynthesis across the transition region where bubble formation commenced because bubble volume and composition could not be measured. Our MIMS technique offers a unique solution to this problem. Because the instrument can determine accurate changes in Ar concentration, it can be used to 'observe' the formation of bubbles. This is possible as the bubbles grow, dissolved gas is repartitioned between the water and gas phase, and with time the dissolved Ar concentration declines as some Ar moves into the bubble. As soon as a decline in Ar concentration is observed, you know you have bubble formation. Fortuitously, the solubility of Ar and O2 are very similar, and we can take this characteristic and use the O2/Ar ratio as a measure of net photosynthesis, whether or not bubbles are present in the system. What appears to be a progressive slowing of the oxygen evolution rate when bubbles form, the change in O2/Ar ratio exhibits linear increases.

Groundwater and Argon Temperatures

In addition to aquatic denitrification, our instrument is used for groundwater denitrification studies with some interesting analytical twists. Nitrate in groundwater can be a serious ecological and human health problem, and that is often a problem under agricultural land. Denitrification can result in significantly (10s %) elevated N2 above solubility where organic rich water enters the groundwater. As with open water systems (see above), the determination of denitrification is from 'open system' measurements. For groundwater which is not in close contact with the atmosphere, the N2 concentration in the absence of denitrification will be related to the average recharge temperature of rainwater, which varies seasonally. The average recharge temperature can be determined from the Ar concentration, and that 'Ar temperature' provides a reference to the N2 concentration during groundwater recharge. The N2 from denitrification is only that N2 above the recharge concentration. The ability of the MIMS to measure precise concentrations (aside from gas ratios) provides a convenient analytical method for determining recharge temperatures and solubility concentrations. The precision of our instrument for dissolved gas concentrations is on the order of 0.1%, which rivals the very best GC measurement (and without the sample processing).

Our Contribution

We developed and described (Kana et al. 1994) the first MIMS instrument capable of high precision N2/Ar ratios in water suitable for detecting the very small changes in N2 concentration associated with sediment denitrification. MIMS was ideally suited because 1) it provided high precision (0.03%) mass spectrometric measurements, 2) it allowed direct measurements on the water (no head-space equilibration), 3) it accommodated small sample size (<10ml), which is required for incubation experiments, 4) it provided a short analysis time (90 s typical) and 5) it provided both gas ratios and single gas concentrations. The vast majority of N2/Ar denitrification studies use a Bay Instruments inlet.

Metabolic Gases in Aquatic and Marine Systems

Our Primary Citation

Our focus has been on measurements of metabolic gases in aquatic and marine systems owing to our academic background. We are not limited to water analyses, and our instruments can be readily configured for the sampling of dissolved gas streams or samples. There are over 300 published articles citing our original publication and the following applications come from that literature as well as selected non-published work.

Original Citation: Kana, T. M.; Darkangelo, C.; Hunt, M. D.; Oldham, J. B.; Bennett, G. E.; Cornwell, J. C. 1994. Membrane Inlet Mass Spectrometer for Rapid High-Precision Determination of N2, O2, and Ar in Environmental Water Samples. Anal. Chem. 66 (23), 4166-4170

Environmental Denitrification

Denitrification converts nitrate (NO3) to nitrogen gas (N2) and it is a critical process in the aquatic and marine nitrogen cycle because it removes nitrogen nutrient that would otherwise  degrade water quality. It is an anaerobic process typically  associated with sediments, hypoxic/anoxic water, groundwater and soils. The challenge has been to measure the very small amount of N2 from denitrification that is added to the high background concentration N2. In practice, measurements in natural environments typically require much better than 0.1% precision to detect Nfrom denitrification when working with hydric or water samples. This precision is not routinely attainable by gas chromatography, but is readily attainable by measuring N2/Ar ratios by mass spectrometry. Argon (Ar) happens to be a convenient basis (standard) for evaluating differences in N2 concentration because it is not biologically reactive.

Aquatic Ecosystem Metabolism and Oxygen Dynamics

Bay Instruments MIMS is an alternative to the previously preferred Winkler technique with the advantage of allowing smaller volume experiments and shorter incubation durations. Oxygen plays a critical role in ecological systems and its  concentration in water can range from zero to supersaturating. There are a number of suitable measurement techniques for dissolved oxygen (e.g. electrodes, Winkler, optodes), but for some environments (e.g. waters with low biological activity, such as the deep sea, open ocean or oligotrophic lakes), very high precision measurements are required to avoid overly prolonged incubations. This is analogous to the problem of measuring denitrification. MIMS offers comparable precision for O2/Ar as it does for N2/Ar (i.e. ~0.03%), which rivals the very best optimized Winkler method. MIMS can make these measurements on much smaller sample volumes than Winkler and is significantly quicker, however.

Sediment Studies

Isotope labeling studies  Another incubation method involves the application of isotopic (heavy) nitrogen (15N) as nitrate. Denitrification will lead to N2 containing the isotope. Denitrification, therefore, can lead to the production of three different masses of N2 (mass 28 as 14N14N, mass 29 as 14N15N, and mass 30 as 15N15N). The so-called 'isotope pairing technique' involves measuring the ratios of masses 28:29:30. This technique, originally developed using isotope ratio mass spectrometry, is now also accomplished using our MIMS.N2/Ar Method  Denitrification associated with sediment is commonly measured using sediment cores with overlying water. The water layer is subsampled, either through time using static core incubations, or by comparing inlet and outlet concentrations in continuously flowing chambers.  Both methods provide denitrification rate measurements. The static core method requires small volume samples to minimize dilution of the headspace water. In addition, the time-course incubations cause changes in O2 concentration, which may affect denitrification activity. Consequently, it is necessary to resolve changes in N2 concentration within a limited range of O2 concentration changes. Nominal precision on the order of 0.03% by our MIMS allows for such measurements.

Going to Sea

Our MIMS instruments have extensive experience operating aboard oceanographic ships. Prior to the founding of Bay Instruments, we were the first to take a MIMS to sea demonstrating the feasibility of getting data on station and in real time, which can have enormous scientific advantages. Mass spectrometers on research ships is fairly common today. There is current interest in understanding whether the oceans are, on balance, producing organic matter or consuming organic matter, of key importance to understanding the role of the oceans in climate change. Where the ocean is clear blue (same with lakes, for that matter), there are few organisms to affect the O2 and CO2 concentrations, and very high precision measurements are required. Our instruments have been used as an alternative to the previously preferred Winkler technique with the advantage of allowing smaller volume experiments and shorter incubation durations.

With modification of the cryotrap temperature, our instrument becomes a pCO2 analyzer which can provide simultaneous measurements of both dissolved O2 and CO2. Nutrient poor, low activity lake waters have been analyzed for CO2 and O2 dynamics using our high precision MIMS. We have to keep in mind that the MIMS method for CO2 does not include the hydrated forms, bicarbonate and carbonate ions, because charged molecules do not pass the membrane.

Open Water Studies

Although the core incubation methods are now standard approaches, open water sampling methods are actively being evaluated because this type of sampling approach integrates larger spatial and temporal scales. The challenge for these studies is to accurately determine the degree of disequilibrium in N2  or N2/Ar, relative to air saturation (i.e. how much additional N2 is in the natural water, relative to what would be there if no denitrification occurred). In many situations (flowing rivers, shallow lakes, etc), the disequilibrium is typically <1% relative to solubility, again requiring the need for high precision and a reliable method for standardizing against air solubility. Our method of preparing dissolved gas standards provides ~0.1% precision on gas (e.g. N2 or Ar) concentrations, which highly resolves temperature effects on solubility. Although these types of measurements are more challenging than for core incubation measurements, the MIMS method has opened new avenues for detecting denitrification in natural waters.

Aquatic Plant & Algae Photosynthesis: The Bubble Problem

Measuring aquatic plant photosynthesis can be problematic because oxygen concentration may rise to the level where small bubbles form on the leaf or on the sediment surface with benthic algae. Conventionally, researchers would either measure the dissolved O2 concentration changes, or the production rate of bubbles from cut leaves. It was not possible to measure photosynthesis across the transition region where bubble formation commenced because bubble volume and composition could not be measured. Our MIMS technique offers a unique solution to this problem. Because the instrument can determine accurate changes in Ar concentration, it can be used to 'observe' the formation of bubbles. This is possible as the bubbles grow, dissolved gas is repartitioned between the water and gas phase, and with time the dissolved Ar concentration declines as some Ar moves into the bubble. As soon as a decline in Ar concentration is observed, you know you have bubble formation. Fortuitously, the solubility of Ar and O2 are very similar, and we can take this characteristic and use the O2/Ar ratio as a measure of net photosynthesis, whether or not bubbles are present in the system. What appears to be a progressive slowing of the oxygen evolution rate when bubbles form, the change in O2/Ar ratio exhibits linear increases.

Groundwater and Argon Temperatures

In addition to aquatic denitrification, our instrument is used for groundwater denitrification studies with some interesting analytical twists. Nitrate in groundwater can be a serious ecological and human health problem, and that is often a problem under agricultural land. Denitrification can result in significantly (10s %) elevated N2 above solubility where organic rich water enters the groundwater. As with open water systems (see above), the determination of denitrification is from 'open system' measurements. For groundwater which is not in close contact with the atmosphere, the N2 concentration in the absence of denitrification will be related to the average recharge temperature of rainwater, which varies seasonally. The average recharge temperature can be determined from the Ar concentration, and that 'Ar temperature' provides a reference to the N2 concentration during groundwater recharge. The N2 from denitrification is only that N2 above the recharge concentration. The ability of the MIMS to measure precise concentrations (aside from gas ratios) provides a convenient analytical method for determining recharge temperatures and solubility concentrations. The precision of our instrument for dissolved gas concentrations is on the order of 0.1%, which rivals the very best GC measurement (and without the sample processing).

Our Contribution

We developed and described (Kana et al. 1994) the first MIMS instrument capable of high precision N2/Ar ratios in water suitable for detecting the very small changes in N2 concentration associated with sediment denitrification. MIMS was ideally suited because 1) it provided high precision (0.03%) mass spectrometric measurements, 2) it allowed direct measurements on the water (no head-space equilibration), 3) it accommodated small sample size (<10ml), which is required for incubation experiments, 4) it provided a short analysis time (90 s typical) and 5) it provided both gas ratios and single gas concentrations. The vast majority of N2/Ar denitrification studies use a Bay Instruments inlet.