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 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 is a critical process that mediates the ecological nitrogen cycle by converting nitrate (NO3–) to nitrogen gas (N2) and therefore removes nitrogen nutrient that could degrade water quality. Within aquatic and marine systems, this microbial process is typically associated with sediments or water with no oxygen. N2 concentration is high, however, due to its solubility with air. The challenge has been to measure the very small amount of N2 from denitrification against the high background concentration. In practice, measurements on natural environments typically require much better that 1% precision to detect N2from denitrification.
One common method involves sediment core incubations whereby the headspace water is sampled for changes in N2 concentration, typically measured as the N2/Ar ratio, where Ar (Argon) is assumed to be constant due to its non-reactivity. The method requires small volume samples to minimize dilution of the headspace water. In addition, the 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% allows for such measurements. 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, may lead to the production of three different masses of N2 (mass 28 as 14N14N, mass 29 as 15N14N, and mass 30 as15N15N). 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 MapOpen water studies Although the core incubation methods are now standard approaches, open water sampling methods are actively being pursued because these types of sampling approaches integrate 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 water relative to what would be there if no denitrification occurred). In many situations (flowing rivers, shallow lakes, etc) the disequilibrium can be <1%, again obviating the need for high precision and reliable methods for standardizing against air solubility. Although these types of measurements are more challenging than for core incubation measurements, the MIMS method has opened up new avenues for detecting denitrification in natural waters.
Ground Water 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, but in areas where there is enough organic matter to support the microbes, denitrification can be substantial (though few measurements have been made) and cause significant reductions in nitrate concentration. The measurement problem relates to the fact that groundwater comes from rainwater that may fall at any time of the year. Because solubility of N2 is temperature dependent (~1.5% change per oC), the N2 concentration in groundwater, purely from solubility, depends on the temperature and amount of rainwater that percolates to the water table. The N2 from denitrification is only that N2 above the solubility concentration. One way of determining the solubility is to use a ‘tracer’ of temperature, in this case, the Ar concentration in the groundwater. Argon is inert and only affected by physical processes (like solubility), so its concentration in groundwater should reflect the solubility equilibrium temperature. That temperature is then used to determine the solubility concentration of N2, which becomes the reference for comparing the measured groundwater gases. One somewhat novel aspect of MIMS is that the measurement signals are proportional to concentration and therefore, MIMS not only provides measures of gas ratios (e.g. N2/Ar) but also N2 and Ar concentrations. Our instruments’ precision for dissolved gas concentrations is on the order of 0.1%, which rivals the very best GC measurement (and without the sample processing).
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 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.
Aquatic Ecosystem Metabolism and O2 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.
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.
Aquatic Plant and Algae Photosynthesis and 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 ration exhibits linear increases.
Denitrification and Metabolic Gases
Working closely with a research group studying groundwater denitrification, there was interest in determining whether gradients in N2/Ar in the soil above the groundwater (vadose zone) were measurable. MIMS (membrane inlet mass spectrometer) was not a practical solution for this problem. Instead, soil gas samples were collected from the field and transported back to the laboratory for high precision analysis of gas ratios. Very high precision was required to resolve the expected very small changes in vadose zone N2/Ar due to the high N2 concentration of soil air.
Our basic MIMS instrument was converted to a capillary gas sampler by simply plugging in a capillary adapter with suitable dimensions to allow the mass spectrometer to sip air. A small sample reservoir (~0.5 cc) with injection septum allowed small volume syringe gas samples to be measured. Precision for N2/Ar was comparable (0.05%) to MIMS measurements. We subsequently developed an alternative injector configuration that avoids precise volumetric injections while maintaining the high precision aspects of the measurements. This has been used for simultaneous O2 and CO2 measurements in studies of microbial respiration.
Calibration and Standardization
Quite a long time ago, we were approached by a company about measuring Ar in water. This proved to be a fruitful collaboration for both parties in that it led to significant improvements in our methods and resulted in technology for this company to calibrate and standardize their aqueous ‘standard’ solutions. They liked our Ar data enough that they challenged us with O2, the real source of their problem. The problem was a slow loss of O2 over a period of weeks inside their calibration standards. The loss was ‘small’, <10%, but it was critical to understand the cause. We helped sleuth it out by providing high precision O2 data that allowed them to compare various treatments.
The Bay Instruments MIMS is now used by this company for production line calibration and standardization and quality control and for laboratory assessment needs. In addition to O2, the instruments are configured to simultaneously measure pCO2. Our instruments have been an integral part of their manufacturing business for the last 10 years.
This project directly led to our present method of calibration and standardization of the mass spectrometer. At that time, we were able to ‘come back to’ the same O2 concentration on samples measured over a period of many weeks at a level better than 0.2%. We do better now as a result of further research on optimization.