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Note 67: Using Chemical Reaction Interface Mass Spectrometry (CRIMS) To Monitor Bacterial Transport In In Situ Bioremediation

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By Mary DeFlaun, Mark Fuller, Allen Thomas, Envirogen, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08536.

By Steven Colby, Eric Butrym, Scientific Instrument Services, 1027 Old York Road, Ringoes, NJ 08551

By Tullis Onstott, Department of Geosciences, Princeton University, Princeton, NJ 08544.

Presented at PittCon 98, Orlando, FL., March 1998

Abstract

The introduction of degradative bacteria into aquifers is becoming an increasingly attractive bioremediation strategy. This is in part due to a significant cost advantage over traditional technologies. Obtaining a uniform bacterial distribution throughout the contaminated zone is pivotal to achieving rapid degradation rates. Tracking the migration of the bacteria injected underground is, therefore, of great interest to those involved in remediation of subsurface environments. Distinguishing the injected bacteria from the indigenous microbial population is often a difficult, time-consuming and hence an expensive process. Tagging the degradative microorganisms with a chemical or isotopic marker is a less costly alternative. This tactic can lead to erroneous results, however, if the marker becomes dissociated from the bacteria through death or predation.

Both of these problems are solved through the use of stable isotope labeling and detection of bacterial-specific lipids. This can be accomplished with the use of Gas Chromatography and Chemical Reaction Interface Mass Spectrometry (GC-CRIMS). The CRIMS technique identifies isotopically labeled species by isotope ratio determination. Chromatographic effluent is mixed with a reactant gas and passed into a microwave plasma. Specific combustion products are produced which permit the measurement of isotope intensities with the interference of isotopologs. This technique provides rapid quantification of the degradative bacterial concentration in subsurface samples at concentrations levels below the detection limit of conventional methods.

Figuare 1

Figure 1 - Schematic representation of a typical bacterial injection for bioremediation of groundwater pollutants. Degradataive bacteria are pumped into the ground water at several depths below the water table via an injection well (red), ofaten mixed with nutrients and air. Tahe bacteria are carried througn conductive areas of the acquifer into the contaminated area (magenta) by the pushing action of the injection well, as well as the pulling action of the removal well(gareen). Monitaoring wells (blue) are used to remove samples for assessing the degradation and movement of the contaminant, as well as the effectiveness of bacterial transport through the contaminated area.

Introduction To Bioremediation and Bacterial Transport

Bioremediation is the use of biological processes to transform and degrade pollutants in the environment. Often times, this is accomplished by developing specific strains of bacteria which are highly effective at metabolizing the target pollutant, and are often engineered to possess other desirable traits as well. One of these desirable traits is a reduced tendency to adhere to sediment surfaces, and hence, a greater ability to be transported to the areas of the subsurface which are contaminated. In order to test potential organisms and monitor the movement of injected organisms during bioremediation activities, it is necessary to "tag" the degradative organisms is some way. This had traditionally been done by engineering the organisms to have a unique antibiotic sensitivity pattern. This method is falling out of favor, however, due to the rise in multiple antibiotic resistant pathogenic bacteria. In the laboratory, another dominant method has been to label the degradative organisms with a radioisotope (14C, 35S, 3H) and measure the radioactivity in the effluent from sediment cores, which allows very small numbers of organisms to be detected. For obvious reasons, this is not an acceptable method for field studies. The use of stable isotopes and GC-CRIMS, however, parallels the use of radioisotopes, and is expected to yield comparable results. When only specific components of degradative bacterial cells (ie., proteins, membrane fatty acids, DNA, RNA) are extracted and analyzed, it will also be possible to verify that any stable isotope observed represents the target organism, rather than a transfer of isotope from the injected bacteria to an indigenous bacteria or protozoa due to cell death or predation.

Introduction To CRIMS

Chemical Reaction Interface/Mass Spectrometry is a selective, sensitive, and versatile technique by which specific isotopes or elements can be monitored. CRIMS parallels the use of radioisotopes in that each technique monitors for elemental tags that are independent of the tags chemical structure or environment. In the CRIMS technique, chromatography effluent is introduced into a high-temperature microwave plasma. The analyte molecules are decomposed and then reacted with a specific reactant gas. This results in a small number of well characterized polyatomic products. These compounds are then detected by mass spectrometry. The generation of new small compounds reduces the possibility of interference from isotopologs and eliminates the large isotopic mass profiles found in larger molecules. The isotope ratios measured in CRIMS can easily be used to identify and quantify the presence of isotope tags. A chromatogram showing only enriched species can therefore be produced. CRIMS has the important advantage that stable isotopes may be used as tags. This eliminates the use of radioactive material and greatly expands the possible applications of isotopic labeling. These applications can be found in pharmacology, geology, and environmental science. The limit of detection for radio labeled material is better with GC-CRIMS than by on-line radio-chromatography.

Figaure 2a

Figure 2a - Chromatogram of qualitative fatty acid methyl ester standard mix as determined by GC-CRIMS analysis. Each component of the standard wa present at an approximate amount of 40 ng.

Figure 2b

Figure 2b - 13C abundance in qualitative fatty acied methyl ester standard mix as determined by GC-CRIMS analysis. Each of the 26 components of the standard were present at an approximate amount of 40 ng. Average 13C abundance was 1.28 +/- 0.08%

Figure 2c

Figure 2c - Chromatogram of fatty acid methyl esters extracted from a pure bacterial culture as determined by GC-CRIMS analysis. 13C abundance ranged from 1.07 to 1.28% with a mean of 1.18%

GC-CRIMS METHODOLOGY

INSTRUMENTATION
HP 5890 Series II gas chromatograph

HP 5971 mass spectrometer

Opthos Instruments microwave power source

DB-5 Capillary column, 30 m x 0.25 mm x 0.5 µm film thickness

GC OPERATING PARAMETERS
Injection Manual, splitless
Sample volume 1-2 µL in pentane
Inlet temperature 250°C
Oven temperature 100°C for 5 min, 10°C/min to 200°C, 4°C/min to 250°C, hold for 10 min
Transfer line temperature 280°C
Carrier gas Ultrahigh purity He at constant pressure of 16.2 psi (45.4 cm/s)
Reactant gas Anhydrous sulfur dioxide
Microwave energy 70 W forward power and <20 W reflected power
MS OPERATING PARAMETERS
Mode Single ion monitoring at 44, 45 and 76 amu (dwell of 500, 500 and 50 ms, respectively)
Solvent delay 5 min
MS temperature ~175°C
Vacuum <8 x 10E-5 torr
MS electrical potential 2200 eMV
DC polarity Positive

Conclusions

-GC-CRIMS is suitable for examining bacterial fatty acid methyl esters, yielding acceptable resolution for peak identification.

-Bacterial fatty acid methyl esters in a standard mixture have an abundance of 13C which is only slightly elevated above reported natural abundance levels of 13C.

-Extractions of membrane fatty acids from a pure bacterial culture yielded sufficient material for GC-CRIMS analysis, and 13C abundance was slightly lower than that observed in the qualitative standard.

Future Work

-Determination of isotopic enrichment in bacterial fatty acids extracted from bacteria grown on 13C-enriched substrates.

-Laboratory studies to monitor transport of isotopically labeled bacteria in saturated sediment cores.

-Field studies to evaluate transport of isotopically labeled bacteria in groundwater.

Selected References

CRIMS: chemical reaction interface mass spectrometry, 1994, Abramson, F. Mass Spectrom. Rev., 13:341-56.

Implementation of the chemical reaction interface mass spectrometry technique on a Hewlett-Packard mass-selective detector. 1994. Song, H., Kusmierz, J., Abramson, F., McLean, M. Am. Soc. Mass Spectrom. 5:765-71.

Improved measurement of stable isotope ratios in gas chromatography/mass spectrometry using the microwave-powered chemical reaction interface for mass spectrometry. 1993. Kusmierz, J., Abramson, F. Biol. Mass Spectrom. 22:537-43.

Selective detection of carbon-13, nitrogen-15, and deuterium Labeled metabolites by capillary gas chromatography-chemical reaction interface-mass spectrometry. 1989. Chace, D., Abramson, F. Anal. Chem. 61:2724-30.

Applications of the reaction interface-mass spectrometer technique to the analysis to selected elements and nuclides from sub-microgram quantities of biological macromolecules and xenobiotics. 1988. Chase, D., Abramson, F. J. Res. Natl. Bur. Stand. (U. S.). 93:419.

Accuracy, reproducibility, and interpretation of fatty acid methyl ester profiles of model bacterial communities. 1994. Haack S., Garchow H., Odelson D., Forney L., Klug M. Appl. Environ. Microbiol. 60:2483-93.

Fractionation of fatty acids derived from soil lipids by solid phase extraction and their quantitative analysis by GC-MS. 1993. Zelles L., Bai Q. Soil Biol. Biochem. 25:495-507.

Preliminary observations on bacterial tranport in a coastal plain aquifer. 1997. DeFlaun, M., Murray, C., Holben, W., Scheibe, T., Mills, A., Ginn, T., Griffin, T., Majer, E., Wilson, J. FEMS Microbiol. Rev. 20:473-87.

Development of an adhesion assay and characterization of an adhesion-deficient mutant of Pseudomonas fluorescens. 1990. DeFlaun. M., Tanzer, A., McAteer, A., Marshall, B., Levy, S. Appl. Environ. Microbiol. 56:112-19.

Alterations in adhesion, transport, and membrane characteristics in an adhesion-deficient pseudomonad. DeFlaun, M., Oppenheimer, S., Streger, S., Condee, C., Fletcher, M. Appl. Environ. Microbiol. Submitted.