Does the presence of water vapor, methane, and formaldehyde over certain regions of the Martian landscape mean that large colonies of methanogenic bacteria still are alive today? Vittorio Formisano, Istituto Fisica Spazio Interplanetario (Rome, Italy) is principal investigator for the Planetary Fourier Spectrometer (PFS) on the European Space Agency's Mars Express orbiter mission. He and his research team presented data in February 2005 that is consistent with the existence of present-day biology on Mars. Speaking to a large audience at the first Mars Express Science Conference that was held at the European Space Research and Technology Center in Norrdjiwk, The Netherlands, Dr. Formisano announced, "We have found methane and formaldehyde in the atmosphere of Mars that match up closely with a water vapor map." To put the discovery in perspective, the lifetime of methane gas in the Martian atmosphere is only 300–600 years and unless there was a source constantly replenishing the gas, it should have dissipated millions of years ago.  

The actual discovery of trace amounts of methane gas (at the 11 ppb level) in the Martian atmosphere was made in 1999 by Vladimir Krasnopolsky, Catholic University of America, Washington, D.C. At the time, Krasnopolsky and his team made the observations using an Earth-based telescope located on Mauna Kea, HI, coupled to a Fourier-transform spectrometer, which placed the upper limit of methane at about 10 ppb. Krasnopolsky then said he thought the probability of the methane having a biological origin was about 70%. However, what makes the Formisano Planetary Fourier Spectrometer team discovery so important is that they have linked methane outgassing from the subsurface of Mars to its association with formaldehyde, which, according to Formisano, is an oxidation by-product of methane. The formaldehyde was found in much greater abundance than the methane, at 130 ppb. Consider, too, that formaldehyde only has a lifetime of 7.5 h in the Martian atmosphere due to untenanted UV light breaking it down and it becomes apparent that something is replenishing it at a very fast rate. The history of formaldehyde (H

CO) on Mars started with a tentative detection using the infrared spectrometer from the Russian Phobos 2 orbiter that operated for only two or three months in 1989.  

 Figure 1. This map shows the concentration of water vapor close to the soil around the equatorial region of Mars. The areas of least concentration are in purple, the highest in green.			

Krasnolpolsky was the principal investigator of the infrared spectrometer on Phobos 2 and he some observed spectral features that indicated formaldehyde with a mixing ratio in the Martian atmosphere of 100–300 ppb. Although he was a coauthor on a scientific paper about this subject (2), Krasnopolsky was skeptical about finding much formaldehyde because it is broken down and removed by the solar UV in only 10 h on Mars. Later, he studied a high-resolution spectrum of Mars taken at the Kitt Peak National Observatory (Kitt Peak, AZ) and established an upper limit to formaldehyde on Mars at about 3 ppb. "I had heard about the detection of formaldehyde by Formisano and his team a few months ago but have not seen their spectra yet. The photochemistry of the Martian atmosphere predicts that formaldehyde is less abundant than methane by a factor of 1,000,000. Their detection means that there is a process that is more effective in the production of formaldehyde than that of methane by a factor of a few million! I don't know any natural process of this kind. I think that methanogenic bacteria are a source of methane on Mars." 

Investigating the Origin of Methane and Formaldehyde

Working out the math to figure out how much methane and formaldehyde is produced, Formisano concluded that 150 tons of methane is being produced each year on Mars, while 2,500,000 tons of formaldehyde are being produced. On Earth, nearly all the methane found in the atmosphere is generated through the actions of microorganisms, which adds up to 500 million tons per year.  

Figure 2. Arabia Terra is one of the three equatorial regions where the PFS instrument detected both water vapor and methane concentration. The other two areas are Elysium Planum and Arcadia Memnonia.			

To make the matter even more interesting for Mars, Formisano studied water vapor maps produced by NASA's Mars Odyssey spacecraft and found that the vast reservoirs of underground water ice had a direct correlation to the location of the methane and formaldehyde. Because the methane was not distributed uniformly in the Martian atmosphere but was found only in areas where water vapor had been detected, was it possible that vast colonies of living microorganisms could be putting it there today? Formisano told 

 magazine in January that he felt the data pointed to life in the soil of Mars. If true, then perhaps the intriguing but ambiguous results from the 1976 Viking Labeled Release biology experiment found life after all. This will be a matter for the scientific community to judge in the near future as new discoveries are made; however, Formisano has weighed three possible explanations for the discovery of so much methane and formaldehyde on Mars. 

 In this scenario, active hydrothermal subsurface regions can have temperatures in excess of 400 °C, where methane then is formed from a reaction with CO

 and water. The methane eventually would outgas through the soil and into the atmosphere. Iron oxides in the soil as the methane rises then could be changed, theoretically, into formaldehyde. However, to date, there is no solid evidence for any active volcanism on Mars.  

Figure 3. This "spectrum" was recorded in the areas where the methane concentration is higher. The black line shows the measured spectrum, compared to the computed methane spectrum (red line) and indicating 35 ppb of methane in the atmosphere. In other areas, the measured average of methane at Mars is only 10 ppb.			

 It is well known that many comets contain abundant methane and organic molecules that are thought to be produced by a reaction of solar UV and cosmic rays bombarding the water ice they contain. Perhaps Mars, like many comets, might have a similar occurrence underway. 

 Mars has abundant microbial life 200–300 m below the soil in large aquifers.  			

The Mars Express Planetary Fournier Spectrometer is a highly sensitive double-pendulum infrared spectrometer specifically designed for atmospheric studies, although it can distinguish mineral compositions on the surface of Mars as well. The spectrometer is divided into two channels — a short wavelength channel and a long wavelength channel, with a resolution of 2 cm

 (2000 spectral points in the LW and 8000 spectral points in the SW channel). It has a spectral resolution of 2 cm

 and covers a wavelength range from 1.2–45 μm. It is well suited for determining the precise composition of the Martian atmosphere and works by analyzing wavelengths of sunlight in the range of 1.2–45 μm that are absorbed by molecules in the atmosphere and from the infrared radiation they emit. 

The spectrometer will measure vertical pressure and temperature profiles of carbon dioxide (which makes up 95% of the Martian atmosphere) and seek water, carbon monoxide, methane, and formaldehyde abundances. For the discovery of methane and formaldehyde in the Martian atmosphere, over 2931 spectra were obtained in 24 orbits of the planet focusing on areas located on and near the equatorial region. The highest concentrations of methane were measured over three large areas on Mars known as Arabia Terra, Elysium Planum, and Arcadia-Memnonia. These regions also were associated with high concentrations of hydrogen indicating water, water vapor, or frozen water ice. 

 is a research associate for the Cardiff Centre for Astrobiology (Cardiff, Wales, U.K.). 

Articles

A report published earlier this year (1) discussed a UV–vis-NIR instrument designed for use on NASA Mars rover missions. This article follows up with coverage of the Planetary Fourier Spectrometer on the European Space Agency's Mars Express orbiter mission.

Spectroscopy and the Search for Ancient Life on Mars, Continued

X-rays are attenuated as they pass through matter. That is, the intensity of an X-ray beam decreases the farther it penetrates into matter. Basically, each interaction of an X-ray photon with an atom of the material removes an X-ray from the beam, decreasing its intensity. 

The amount of decrease in intensity of the X-ray beam depends upon two factors: 					

A characteristic of the material called its "absorption coefficient" (

The intensity decreases exponentially with the distance traveled, or

 is the initial X-ray beam intensity. Note that this exponential decay of photon intensity applies in the optical region of the electromagnetic spectrum as well. In this region, it is known as the Beer–Lambert law.  

 absorption coefficient. Note that the term μ , by conventional nomeclature, is often referred to as the 

. These two coefficients are related by the density of the material (ρ) as μ = 

An interesting application of this equation is to determine the depth of penetration of X-rays. The attenuation length is defined as 

the depth into the material where the intensity of the X-rays has decreased to about 37% (1/

, sometimes called Euler's number or Napier's constant, is the base of natural logarithms, or 

 ≈ 2.7183.] Then, substituting into Equation 1, we get 

This also is referred to as the "mean free path" of the X-rays. 

Cd radioisotope source of X-rays with an energy of 22.1 keV, how far do these penetrate a piece of pure iron, (atomic number 26)? From Table I, we find the mass absorption coefficient for iron at 22.1 keV is μ = 18.2 cm

 = 0.007 cm = 0.07 mm = 70 μm. A comparison of this depth for the same incoming X-ray energy both for lighter and heavier elements is shown in Table I. 

Measuring the Mass Absorption Coefficient

Figure 1: Attenuation of X-ray intensity. 

We need a source of X-rays — the radioisotope Cd-109 or an X-ray tube, for example — and a detector such as a Geiger counter. Place a piece of foil of thickness 

 between the source and detector and measure the radiation intensity 

, as shown in Figure 1. Now place a second piece of foil with a thickness 

 in the path and measure the new intensity 

For example, we use an X-ray tube run at 35 kV and a 0.025-mm thick piece of copper foil. With one foil thickness, our Geiger counter measures 185,000 counts per minute (cpm). When the foil is doubled and the measurement repeated, we find a count rate of about 155,000 cpm. The density of copper is 8.94 g/cm

The accepted value for the mass absorption coefficient of copper at this energy is approximately 7.0 cm

/g. This is a reasonable agreement given the rather crude experimental set-up in which the Geiger counter used had a low-resolution analog readout meter. 

When a sample contains more than one component, the total mass absorption coefficient is the sum of the individual coefficients, each multiplied by the weight fraction present. That is,   

Consider an alloy containing 70% Ni and 30% Mo. What is the mass absorption coefficient of this sample at an excitation energy of 22.1 keV (

Mo)(μMo) = (0.7)(23.8) + (0.3)(61.9) = 35.3 cm

The mass absorption coefficient of a material is energy dependent. In general, this coefficient decreases with increasing energy of the penetrating X-rays. Figure 2 shows a plot of the mass absorption coefficient for molybdenum from about 7 to 35 keV. 

Figure 2: Mass absorption coefficient of molybdenum.  

Note, however, that we do observe sharp increases in this coefficient at certain energies. These discontinuities occur at the minimum energies for excitation of a particular electron shell, that is, at the binding energies. For example, in Figure 2, we find a discontinuity at about 20 keV. This is the K-shell electron binding energy.  			

These discontinuities in the mass absorption coefficient are called "absorption edges." After an absorption edge, the general decrease with increasing energy continues. 

Figure 3 presents the energy dependence of the mass absorption coefficient of Molybdenum in the more traditional log–log format. Also evident are the L absorption edges at about 2.5, 2.6, and 2.9 keV. These correspond to the electron binding energies of the three L-shell subshells.  

Figure 3: LogÃ¢ÂÂlog plot of mass absorption coefficient of molybdenum. 			

The attenuation of X-rays by matter is a specific example of the more general phenomenon of the attenuation of electromagnetic radiation by matter. In the optical region of the E–M spectrum, this phenomenon finds significant spectrometric application in the form of the Beer–Lambert law.   

In this X-ray tutorial, the authors attempt to answer the frequently asked question, "How deep do the X-rays penetrate my sample?"

Tutorial: Attenuation of X-Rays By Matter

Over the past decade, Fourier-transform infrared microspectroscopy (IMS) has emerged as a tool to study plant growth and development at the molecular level. Synchrotron radiation (SR) as a source for IMS has offered improvements in studying plant samples with limited light throughput. The small effective source size and high brightness of the synchrotron beam also allows study of samples with small areas with spatial resolution to the diffraction limit of 5–15 μm. However, the use of SR-IMS to study plant structures is still in its infancy. There are a limited number of research groups that use IMS to study plant microstructures with McCann's (1, 2) group at Purdue, and Schreiber et al. (3, 4) at Universität Würzburg in Germany, leading the way. Only a few scientists have used synchrotron facilities for this type of research. Raab et al. (5, 6) studied biological and chemical changes in growing legume roots at the Advanced Light Source (ALS) of Lawrence Berkeley National Laboratory, Berkeley, California. Wetzel et al. (7) and Yu et al. (8, 9) studied the microstructures of wheat and barley seeds, respectively, at the National Synchrotron Light Source (NSLS) of Brookhaven National Laboratory, Upton, NY. The results of these studies suggest a potential use of SR-IMS to analyze changes in plant structures due to contact with organic contaminants, commonly called herbicides. 

The main focus of our research is studying the fate and transport of organic contaminants in plants. Phytoremediation, the use of plants to remove contaminants from water, soil, or air, has grown greatly over the past 20 years. Nevertheless, an effective technique to determine the fate of organic contaminants directly in plants is needed. Typical extraction methods degrade plant tissues or must be applied to ground-up tissue, while most radiolabel techniques cannot pinpoint the anatomical region in which the contaminants are ultimately bound or transformed. SR-IMS provides a direct alternative to these techniques, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. For herbicides, the tolerable levels can to be too low for direct detection, but the effects of the compound on plant structures might be of interest. In the case of many common contaminants, a several hundredfold concentration of the contaminant accompanies its uptake into a plant with the transpiration stream. 

Currently, our lab is using SR-IMS to study the fate and transport and developmental effects of common aromatic and heterocyclic organic contaminants in sunflower (

) plants. Here we will report on the fate and effects of 2,6-dinitrotoluene (2,6-DNT), an industrial byproduct of TNT production, on maize plants. 

Pioneer Maize seeds (hybrid 32M38) were germinated for three days in vermiculite moistened with sterilized tap water and then transferred to 50-mL centrifuge tubes containing full-strength Hoagland's solution for a 24-h period. Hoagland's solution was prepared as previously reported by Castro et al. (10). The maize seedlings then were distributed into stratified groups with each group having a similar mean weight. The seedlings were grown hydroponically in full-strength Hoagland's solution containing 0, 5, 10, and 15 mg/L 2,6-DNT for a one-week period under continuous lighting. Three plants were used for each treatment group. After the growth period, the maize plants were removed from their treatment solutions and rinsed briefly with deionized water before dissection and cryomicrotome sectioning. The remaining treatment solutions were kept for analysis by high performance liquid chromatography (HPLC). The representative secondary roots from each treatment were pooled (~6 roots/treatment) and samples were cut into 1-cm segments from the tip. The root tissues were frozen onto specimen blocks surrounded by Tissue-Tek OCT (Sakura Finetek USA, Inc., Torrance, CA) at –40 °C, in preparation for sectioning at –20 °C.  The 4-μm thick frozen sections were thaw mounted onto infrared-reflecting "E" glass microscope slides (Smiths Detection, Danbury, CT). The slides then were stored at room temperature until analysis. 

The amount of solution used by the seedlings was calculated, while measuring contaminant uptake by the maize plants was performed using an Altex liquid chromatograph Model 330 attached to a spectrophotometer flow cell (Altex/Hitachi model 100-40) set up for detection at 275 nm. The separation was through a Hamilton (Reno, NV) PRP-1 column (150 × 4.1 mm) with an eluent consisting of methanol:water (9:1) at a flow rate of 1.0 mL/min. Loss of 2,6-DNT from the residual treatment solutions was observed, with 100%, 53%, and 16% depletion at the 5, 10, and 15 mg/L concentrations, showing degradation and uptake by the maize. Water uptake varied with treatment. 

Synchrotron radiation Fourier-transform infrared microspectroscopy (SR-IMS). 

SR-IMS was conducted at the NSLS at Brookhaven National Laboratory at beamline U2B using a Magna 860 spectrometer (Thermo Nicolet Instruments, Madison, WI) coupled with a Nicolet NicPlan IR microscope with a custom-made external cryostat chamber. An in-depth description of Beamline U2B along with a schematic can be found in a report authored by Marinkovic et al (11). The narrow-range, internal mercury cadmium telluride (MCT/A) detector was used, covering a range of 4000–650 cm

. All spectra were collected using the 32× IR objective (Thermo-Spectra-Tech, Shelton, CT) in the reflection mode. The reference spectrum was collected from an unused portion of the low "E" slide at 128 scans with 4-cm

 resolution. The spectra were recorded in absorption mode at a resolution of 4 cm

 with 64 scans co-added to reduce signal-to-noise ratio (S/N). A 30-μm square aperture was used for scouting potential specimens for mapping and taking individual spectra. A 12-μm square aperture with 10-μm steps was used for generating an IR map of root sections using the Atlμs, a component of the Omnic 6.0 software package (Thermo Electron, Madison, WI).  

These spectra are diffraction limited; the plant contains interesting smaller features. 

Radiolabeling of organic contaminants is the method of choice for studying the fate and transport of organic contaminants in plant tissues for most environmental scientists. However, many compounds are not available in isotopically labeled form, and the microstructures of the plant are destroyed during the extraction processes used to determine the location of radioactivity. This limits determining the spatial location and distribution of the organic contaminants. The chemical imaging ability of SR-IMS provides a direct method of pinpointing which plant structures are changed due to exposure and the anatomical region where the organic compound ultimately can be bound or transformed.   

The chemical image produced from SR-IMS uses different colors to represent chemical group intensities generating a false color intensity map. Figure 1b shows the distribution of nitro compounds in root tissue of a maize plant treated with 10 mg/L 2,6-DNT by taking the ratio of peak heights at 1550 (amide II band) and 1530 cm

. The mapped area covered a region of 290 × 70 μm or 29 × 7 pixels. Raw spectra from the vascular region of 10 mg/L 2,6-DNT treated root tissue in Figure 1e shows the peaks used to generate the peak height ratio map. Blue and green colors signify high intensities, while red and white show little or no presence of the nitro groups. The intensity range extends from 0 to 175 (Figure 1c), whereas a comparable control map would have a range of 0–0.04 without any distinguishing features. The epidermis and vascular region of the root tissue display the highest distribution of nitro groups compared to little or no distribution in the cortex. This provides evidence that 2,6-DNT may be incorporated into the lignin portion of the plant because lignin is predominantly found in the epidermis and vascular region of the root.  Similar results have been observed in studies of sunflower plants treated with 1H-benzotriazole (13).  

 Figure 1.  False color intensity map of ratio of peak heights at 1550 and 1530 cm-1, showing the distribution of nitro groups in 2,6-DNT treated maize root tissue at each pixel. (a) Visual image of maize root cross section approximately 500 Î¼m from the tip. The blue rectangles in the visual images represent the mapped area of 29 3 7 pixels. The black bars represent a length of 100 Î¼m. e = epidermis, c = cortex, v =  vascular region; (b) chemical image; (c) chemical intensity ruler; (d) 3-D image; and (e) raw spectra of untreated and 10 mg/L 2,6-DNT treated root vascular tissue.			

Spectra taken of the cortex of untreated, 5 mg/L 2,6-DNT, and 10 mg/L 2,6-DNT treated maize root tissue were very similar although there are important differences in ratios of several peaks (Figure 2b). Spectra from 15 mg/L 2,6-DNT treated root tissue are not shown because the root tissues were dark and badly damaged, making it difficult to obtain quality spectra from these samples. There does not appear to be a large decrease in the protein (1550 and 1650 cm

) regions of the treated tissue, and there are no strong bands indicative of the presence of 2,6-DNT. The dominance of the nitro group peaks at 1530 cm

 can be seen in Figure 2a, which shows the spectrum of 2,6-DNT. The presence of the 1530 cm

 peak can be seen in the typical spectra of vascular tissue from maize plants treated with 2,6-DNT (Figure 2c) as a large shoulder on the 1550 cm

 amide II peak. The obvious overall increase in absorption over the whole 1500–1600 cm

 region but not in the carbohydrate region (1200–1000 cm

) does suggest that the changes are due to treatment with 2,6-DNT and not due changes in section thickness or denser tissue. In some cases, the 1530 cm

 peak might be higher in absorption than the amide II peak at 1550 cm

, as seen in Figure 1e. Bands due to the aromatic ring of 2,6-DNT appear in the treated samples at 820–975 cm

 is indicative of C–C ring stretch followed by a ring H–C–C bend near 842 cm

. Peaks due to the methyl group of 2,6-DNT are at 895 cm

. Decreases in the carbohydrate region and changing ratio of protein bands indicate that the uptake of 2,6-DNT by the plant might have altered their production. Detailed interpretation of these spectral changes requires use of principal components analysis or similar discriminating technique (14 ).  

Figure 2. (a) Spectrum of 2,6-DNT prepared in a 1% (w/w) pellet of KBr; (b) typical spectra of cortex of root tissue from untreated, 5 and 10 mg/L 2,6-DNT treated maize plants; (c) typical spectra of vascular region of root tissue from untreated, 5 and 10 mg/L 2,6-DNT treated maize plants. Each example spectrum is an average of 20 spectra from their corresponding region.			

SR-IMS was used to determine the distribution of 2,6-DNT in maize root tissue. Chemical imaging of the root tissue showed high levels of the compound in epidermis and vascular tissue, indicating that 2,6-DNT can be found in the lignin fraction. TNT is known to bind predominantly to the lignin fraction of wheat plants (15). The presence of the nitro peak at 1530 cm

 peaks, and bands associated with the aromatic ring that appear in the 820–975 cm

 suggest that 2,6-DNT was not fully transformed upon incorporation. Loss of the 1350 cm

 band, however, shows that the compound is modified. No spectra are available for likely products such as aminonitrotoluene or hydroxyaminonitrotoluene. Limited metabolic capacity might explain why concentrations higher than 10 mg/L kill maize plants. We were able to demonstrate that SR-IMS can be used as a direct technique to determine fate and effects of organic compounds in plant tissue.  

Funding for experimentation at the NSLS at Brookhaven National Laboratories was provided by the Department of Energy. Further support was provided by the U.S. Environmental Protection Agency under assistance agreement R-825550 through the Great Plains/Rocky Mountain Hazardous Substance Research Center and the Kansas Agricultural Experiment Station (contribution number 06-32-J), both in Manhattan, KS. The authors also would like to thank the staff at the NSLS at Brookhaven National Laboratories and the Kansas State University Veterinary Diagnostic Laboratory, Manhattan, KS, supervised by Cindy Chard-Bergstrom, for the use of their cryomicrotome.  

 are with the department of biochemistry at Kansas State University (Manhattan, KS). 

 is with the Albert Einstein Center for Synchrotron Biosciences, Beamline U2B, National Synchrotron Light Source, at Brookhaven National Laboratory (Upton, NY). E-mail: 

1. M.C. McCann, M. Bush, D. Milioni, P. Sado, N.J. Stacey, G. Catchpole, M. Defernez, N.C. Carpita, H. Höfte, P. Ulvskov, R.H. Wilson, and K. Roberts, 

7. D.L. Wetzel, A.J. Eilert, L.N. Pietrzak, S.S. Miller, and J.A. Sweat, 

8. P. Yu, J.J. McKinnon, C.R. Christensen, and D.A. Christensen, 

9. P. Yu, J.J. McKinnon, C.R. Christensen, D.A. Christensen, N.S. Marinkovic, and L.M. Miller, 

10. S. Castro, L.C. Davis, and L.E. Erickson, 

Practic. Period. Hazard. Toxic Radioact. Waste Managem

11. N.S. Marinkovic, R. Huang, P. Bromberg, M. Sullivan, J. Toomey, L.M. Miller, E. Sperber, S. Moshe, K.W. Jones, E. Chouparova, S. Lappi, S. Franzen, and M.R. Chance, 

12. N.S. Marinkovic, M.R. Chance, K.M. Dokken, L.C. Davis, D.H. Linkous, and J.M. Flinn, 

13. K.M. Dokken, L.C. Davis, L.E. Erickson, S. Castro-Diaz, and N.S. Marinkovic, 

14. B.A. Budevska, S.T. Sum, and T.J. Jones, 

15. C. Sens, P. Scheidemann, and D. Werner, 

The fate and transport of organic contaminants and their impact upon plant development has been an important topic in environmental science. Here the authors report the use of synchrotron radiation Fourier-transform infrared microspectroscopy (SR-IMS) as a direct method for monitoring the fate and effects of 2,6-dinitrotoluene (2,6-DNT) in maize (Zea mays L.) root tissue.

Using SR-IMS to Study the Fate and Transport of Organic Contaminants in Plants

Columns

Previous methods for linearity testing discussed in this series contain certain shortcomings. In this installment, the authors describe a method they believe is superior to others.

Spectroscopy-09-01-2005

Spectroscopy and the Search for Ancient Life on Mars, Continued

Tutorial: Attenuation of X-Rays By Matter

Using SR-IMS to Study the Fate and Transport of Organic Contaminants in Plants

Chemometrics in Spectroscopy - Linearity in Calibration: How to Test for Non-linearity